Small molecule inhibitors of protein tyrosine phosphatases and used thereof

ABSTRACT

Small molecule compounds derived from α-sulfophenylacetic amide (SPAA) are provided as novel sulfonic acid based pTyr mimetics. These compounds effectively inhibit a variety of protein tyrosine phosphatases (PTPs), such as mPTPA, mPTPB, LMWPTP, and Laforin. Use of these compounds as pharmaceutical agents for treating diseases associated with abnormal protein tyrosine phosphatase activity is also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/169,148 filed Jun. 1, 2015 and U.S. Provisional Application No.62/169,122 filed Jun. 1, 2015, both of which are hereby incorporated byreference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH & DEVELOPMENT

This invention was made with government support under CA152194 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND OF THE DISCLOSURE

The field of the disclosure relates generally to small moleculeinhibitors of protein tyrosine phosphatases (PTPs). Protein tyrosinephosphorylation is a key post-translational modification used byeukaryotic organisms to control protein function. Proper levels oftyrosine phosphorylation are maintained by protein tyrosine kinases(PTKs) and protein tyrosine phosphatases (PTPs). Disturbance of thecoordinated and opposing activities of PTKs and PTPs leads to aberranttyrosine phosphorylation, which is associated with the development andpathogenesis of numerous human diseases including cancer, diabetes andautoimmune disorders. Accordingly, dysfunctional tyrosinephosphorylation mediated signaling networks present enormousopportunities for therapeutic intervention. This has prompted thedevelopment of inhibitors for disease-associated PTKs, over two-dozen ofwhich have already been approved for clinical use.

For example, mPTPB, a virulence factor of the mycobacterium tuberculosis(Mtb) strain, is a novel drug target of tuberculosis (TB). Tuberculosiscontinues to be a leading epidemic in the world which killsapproximately 2 million and infects 9 million people annually (WHOReport, 2010 on Global TB Control, 2010). The present short course chemotherapy, formulated 40 years ago, starts with administration ofisoniazid, pyrazinamide, and rifampicin for two months, and thenisoniazid or rifampicin for four months, which is time-consuming,tedious and costly. The emergence of multidrug-resistant (MDR),extensively resistant (XDR), and HIV-associated TB has also severallychallenged current therapies (Harries et al., Lancet 2010, 375,1906-1919; Gandhi et al., Lancet 2010, 375, 1830-1843). With thecurrently available approaches, it is impossible to eliminate the TBdisease as a worldwide threat by reducing incidence below one case permillion populations by 2050, a long term target of MillenniumDevelopment Goals (Marais et al., Lancet 2010, 375, 2179-2191). In thisregard, molecules targeting mPTPB provide a solution for thesechallenges.

mPTPA is another PTP (17.5 kDa) secreted by the Mtb strain, which has38% sequence and large overall structural similarities with human lowmolecular weight PTPs (LMPTP). It has been found that the expressionlevel of mPTPA in M. bovis BCG increased upon entry into stationaryphase in vitro or upon infection of human monocytes, implying a positiverole of this enzyme during infections. mPTPA is also capable ofinhibiting phagocytosis and increasing actin polymerizations inmacrophages. Human mPTPA, when combined with its substrate (VPS33B),inhibits phagosome-lysosome fusion, a process arrested in Mtb'sinfection. A genetic deletion of mPTPA attenuated Mtb growth in humanmacrophages. These findings indicate that mPTPA is another potentialtarget for the development of novel anti-TB agents, in addition tomPTPB. So far, only few mPTPA inhibitors have been reported, and most ofthem lack potency and specificity over human PTPs. The compounddeveloped by Ellman (Rawls et al., Bioorg. Med. Chem. Lett. 2009, 19,6851-6854) based on phosphonodifluoromethyl phenylalanine (F2PMP) as aphosphotyrosine (pTyr) mimetic, with a Ki value at 1.4 μM, is 11-foldselective for mPTPA over LMPTP, and is 70-fold selective for severalmammalian PTPs such as PTP1B, TcPTP, CD45.

LMWPTP is a positive regulator of tumor onset and progression probablyby dephosphorylating ephrin A2 (EphA2) receptor tyrosine kinase.Clinically, elevated LMWPTP mRNA and protein level have been observed inmalignant samples of breast, colon, bladder, and kidney. LMWPTP is alsoa key negative regulator of insulin signaling, and inhibition of LMWPTP(e.g. by antisense oligonucleotide (ASO) in cultured mouse hepatocytes,liver and fat tissues of diet-induced obese (DIO) mice and ob/ob mice)leads to increased phosphorylation and activity of key insulin signalingintermediates, including insulin receptor-β subunit,phosphatidylinositol 3-kinase, and Akt. Recently, LMWPTP is shown to bea good drug target for cancer and type 2 diabetes Accordingly, smallmolecule inhibitors of LMWPTP are potential treatments for cancer,insulin resistance, type 2 diabetes and obesity. Unfortunately, thedevelopment of LMWPTP inhibitors has met with little success. Moderatelyactive LMWPTP inhibitors have been reported, but these compounds alsoinhibit other PTPS, such as PTP1B, TcPTP, PTPβ.

Laforin is a dual specificity phosphatase encoded by the EPM2A gene, themutation of which has been found in patients with Lafora disease, afatal autosomal recessive genetic disorder characterized by theexistence of inclusion bodies (Lafora bodies) in neurons, heart, liver,muscle, and skin. Patients with this disease usually die within 10 yearsof showing symptoms and do not live beyond the age of 25. Currentlythere is no cure for the disease. It is proposed that this disease iscaused by the mutation in Laforin's carbohydrate binding domain (CBD),making Laforin unable to locate its substrate and eventually leading tothe formation of insoluble polyglucocans, the main component of Laforabodies. Remarkably, whether PTP activity is involved in the developmentof Lafora disease is one of the open questions in the field, and aspecific Laforin inhibitor can provide useful insight.

Further, the Src homology 2 (SH2) domain containing protein tyrosinephosphatase-2 (SHP2), encoded by the PTPN11 gene, has generatedconsiderable interest as an oncology target. Biochemically, SHP2 servesas a positive signal-transducer downstream of most, if not all, receptorPTKs and is required for Ras-ERK1/2 cascade activation. Consistent withits oncogenic potential, germline gain-of-function mutations in PTPN11cause Noonan syndrome, whereas somatic activating PTPN11 mutations occurin juvenile and adult myeloproliferative diseases and contribute toseveral types of solid tumors including lung adenocarcinoma, colon andprostate cancer, neuroblastoma, glioblastoma, melanoma, andhepatocellular carcinoma. SHP2 is also shown to play a critical role inboth triple-negative and HER2⁺ breast cancer. Finally, given theessential role of SHP2 in growth factor signaling, thwarting SHP2 actionmay also prove effective for cancers caused by abnormal activation ofreceptor PTKs. These findings have spurred an intense effort to developSHP2 inhibitors for novel anti-cancer agents.

Small molecule inhibitors of PTP are invaluable in elucidating themechanisms of these diseases and in providing novel therapeuticinterventions. However, as discussed above, the development in this areahas been largely hurdled by the challenges of developing potent,selective and bioavailable, or simple drug-like PTP inhibitors. Theunderlying reasons are that more than 100 PTPs identified to dateutilize a common catalytic mechanism, and that their highly positivelycharged active sites share a high level of similarity. Targeting anactive site with negatively charged phosphotyrosine (pTyr) substratemimetics, and surrounding regions with additional fragments is a majorstrategy having a certain degree of success. pTyr mimetics play a vitalrole in this regard, and serve as foundations for the development ofpotent and specific PTP inhibitors. This is well demonstrated by F2PMP,a non-hydrolyzable pTyr mimetic designed nearly two decades ago that ledto the discovery of many PTP1B inhibitors to date (FIG. 1).

Carboxylic acid is another typical class of pTyr mimetic that has beenstudied extensively. Specifically, salicylic acid is a novel, cellpermeable pTyr mimetic discovered recently, from which specificinhibitors against LYP, SHP2, and mPTPB have been developed.Consequently, one would consider sulfonic acids as pTyr mimetics inaddition to phosphonic and carboxylic acids. Unfortunately, research inthis subject had obtained very limited success, with scarce reports onseveral moderately active and nonspecific PTP inhibitors.

Accordingly, there is a need in the art to provide small moleculeinhibitors of PTPs, and more particularly, there is a need for highlypotent and specific bioavailable inhibitors of several distinct PTPs,including mPTPA, mPTPB, LMWPTP, Laforin, SHP2, LYP, and HePTP.

SUMMARY OF THE DISCLOSURE

The present disclosure generally relates to small molecule inhibitors ofPTPs and the use of these inhibitors as therapeutics for variousdiseases associated with abnormal protein tyrosine phosphatase activity.

In one aspect, the present disclosure provides a compound of Formula 1a:

or a therapeutically suitable prodrug thereof or a therapeuticallysuitable salt thereof, wherein R₁ is hydrogen and R₂ is selected fromC₁-C₁₀ alkyl, aryl, heteroaryl, —NH—R_(2a), —(CH₂)_(m)NH—CO—R_(x), and—(CH₂)_(n)—R_(2b)—(CH₂)_(q)—NH—CO—CO—NH—R_(y);

-   -   wherein, when R₂ is aryl or heteroaryl, R₂ is optionally        substituted with one or more substituent selected from the group        consisting of C₁-C₄ alkyl, C₁-C₄ alkoxy, C₁-C₄ alkoxy carbonyl,        amino, aryl, benzyloxy (—OBn), —CF₃, carboxy, halogen,        1-imidazolyl, 4-morpholinyl, and nitro;    -   wherein m, n, and q independently are 0-4;    -   wherein R_(2a) and R_(2b) independently are aryl;    -   wherein R_(x) and R_(y) independently are aryl or heteroaryl,        and the aryl or heteroaryl are independently optionally        substituted with one or more substituent selected from the group        consisting of C₁-C₄ alkyl, benzoyl, benzyl, benzyloxy (—OBn),        phenyl, halogen, 1H-benzimidazole-2-yl, and 2-thiophenyl;

-   or wherein R₁, R₂, and the N atom to which they are attached are    joined together to form a monocyclic or bicyclic heterocycle;

-   wherein R₃ is hydrogen or halogen; and

-   wherein R₄ is hydrogen or aryl, the aryl being optionally    substituted with one or more substituent selected from the group    consisting of halogen, C₁-C₄ alkyl, C₁-C₄ alkoxy, phenyl, nitro,    cyano, and —COCF₃.

In another aspect, the present disclosure provides a compound of Formula2:

-   wherein R₂′ is heterocycle, optionally substituted with one or more    substituent selected from the group consisting of C₁-C₄ alkyl, C₁-C₄    alkoxy, C₁-C₄ alkoxy carbonyl, amino, aryl, benzyloxy (—OBn), —CF₃,    carboxy, halogen, 1-imidazolyl, 4-morpholinyl, and nitro;-   wherein R₃′ is hydrogen or halogen; and-   wherein R₄′ is hydrogen or aryl, the aryl being optionally    substituted with one or more substituent selected from the group    consisting of halogen, C₁-C₄ alkyl, C₁-C₄ alkoxy, phenyl, nitro,    cyano, and —COCF₃.

In another aspect, the present disclosure provides a method ofinhibiting a protein tyrosine phosphatase (PTP) selected from the groupconsisting of mPTPA, mPTPB, low molecular weight PTP (LMWPTP), andLaforin in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of thecompound of Formula 1a, a therapeutically suitable prodrug thereof, or atherapeutically suitable salt thereof, or the compound of Formula 2.

In another aspect, the present disclosure provides a method of treatingvarious diseases associated with abnormal protein tyrosine phosphataseactivity, such as tuberculosis, cancer, Lafora disease, and type 2diabetes, the method comprising administering to the subject atherapeutically effective amount of the compound of Formula 1a, atherapeutically suitable prodrug thereof, or a therapeutically suitablesalt thereof, or the compound of Formula 2.

In yet another aspect, the present disclosure provides a pharmaceuticalcomposition comprising a compound of Formula 1a, a therapeuticallysuitable prodrug thereof, or a therapeutically suitable salt thereof, orthe compound of Formula 2 and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the development of PTP inhibitors that rely on the designof pTyr mimetics.

FIG. 2 depicts the crystal structure of SHP2-bound cefsulodin andα-sulfophenylacetic amide (SPAA) as a novel pTyr mimetic.

FIGS. 3A-3C depict three libraries between α-sulfophenyl acetic acid andamines under peptide coupling conditions. FIG. 3A depicts a library inwhich SPAA was attached to diamine linkers and then coupled with 192carboxylic acids to give a library of 960 compounds. FIG. 3B depicts alibrary in which SPAA with various substituents were coupled with 192amines to give a library of 3264 compounds. FIG. 3C depicts a library inwhich SPAA was attached to oxalyl linkers and then coupled with 192amines to give a library of 960 compounds.

FIG. 4 is a scheme depicting the synthesis of precursors forconstruction of the library of FIG. 3A.

FIG. 5 is a scheme depicting the synthesis of precursors forconstruction of the library of FIG. 3B.

FIG. 6 is a scheme depicting the synthesis of precursors forconstruction of the library of FIG. 3C.

FIG. 7 depicts Compound L335-M34 as a reversible and competitiveinhibitor of mPTPA with pNPP as a substrate. Lineweaver-Burk plot forL335-M34-mediated mPTPA inhibion. Compound L335-M34 concentrations were0 (●), 50 (∘), 100 (Δ), 150 (▾), and 200 nM (▪). The K_(i) value of56±2.0 nM was determined from three independent measurements.

FIG. 8 depicts the pharmacokinetic profile of lead mPTP inhibitors inguinea pig plasma. Concentration of mPTP inhibitors in the plasma overtime. L335-M34 (mPTPA) 50 mg/kg or L01-Z08 (mPTPB) 20 mg/kg was givenorally once alone or in combination, and blood samples were collected atthe indicated intervals relative to dosing. N=3 per time point; graphrepresents mean±SD values.

FIG. 9 depicts the average guinea pigs body weights during treatment.Numbers after each drug refer to doses (mg/kg). HRZ: Isoniazid (H),60/Rifampin (R), 100/Pyrazinamide (Z), 300; mPTPA/A: L01-Zo8, 20;mPTPB/B: L335M34, 50; n=5 per time point; graph represents meanvalues±SD.

FIGS. 10A-10C depict the activity of adjunctive mPTPA and mPTPBinhibitors against chronic TB infection in guinea pigs. Animals wereinfected vi aerosol with ˜10² CFU of M. tuberculosis H37Rv and wereeither left untreated or were treated with drugs beginning 4 weeks afterinfection. Log₁₀ CFU in the lungs are shown after 2 (FIG. 10A), 4 (FIG.10B), and 6 (FIG. 10C) weeks of treatment. No drug=untreated, HRZ:isoniazid (H), 60/Rifampin (R), 100/pyrazinamide (Z), 300; mPTPA/A:L335M34, 50; mPTPB/B: L01-Z08, 20; numbers after each drug refer todoses (mg/kg); n=4 guinea pigs per time point; *p<0.05, **p<0.01,***p<0.001, HRZ versus HRZ+A+B.

FIGS. 11A-11C depict the effects of drug treatment on body weight (FIG.11A), lung weight (FIG. 11B), and spleen weight (FIG. 11C) in M.tuberculosis-infected guinea pigs. No drug=untreated, HRZ: Isoniazid(H), 60/Rifampin (R), 100/Pyrazinaimde (Z), 300; A: L01-Z08, 20; B:L335M34, 50; n=4/5 per time point. Numbers after each drug refer todoses (mg/kg). n=4 per time point.

FIG. 12 depicts lung inflammation 6 weeks after initiation of treatment.Results are represented as percentage of lung surface area involved,calculated using ImageJ software. Compared to RHZ, RHZ+A/RHZ+A+B aremore effective in reducing lung lesion size and number in M.tuberculosis-infected guinea pigs at month 1.5 after treatment; *p<0.05, HRZ versus HRZ+A/HRZ+A+B.

FIG. 13 depicts the combination regimes significantly reduce lung lesionsize and number in M. tuberculosis-infected guinea pigs at month 1.5after treatment. Low-power (2× magnification) view of hematoxylin andeosin-stained sections of lungs from individual animals that bestrepresents the mean values of 4 animals.

FIG. 14 depicts the chemical structures of cefsulodin and severalrelated β-lactam antibiotics. Cefsulodin is composed by 3 parts:sulfonic acid head group A, β-lactam core B, and isonicotinamide tail C.Moxalactam is the only compound with all 3 parts, carbenicillin andsulbenicillin have A and B parts, cefalonium and cefamandole have partsB and C, while cefdinir, cephalexin and penicillin G just have part B,β-lactam core.

FIG. 15 depicts Lineweaver-Burk plot for cefsulodin-mediated SHP2inhibition as analyzed in Example 9. Cefsulodin is a competitiveinhibitor of SHP2 with Ki at 6.6 μM. Cefsulodin concentrations were 0(Δ), 20 (∘) and 40 (●) μM.

FIGS. 16A & 16B depict LC-MS characterization of SHP2 inhibition bycefsulodin as analyzed in Example 9. LC-MS showed that cefsulodin is notinhibiting SHP2 by covalent modification. FIG. 16A: Under inhibitionassay conditions (pH 7 and 25° C., 20 nM SHP2, 3 mM pNPP, 100 μMcefsulodin, 50 mM 3,3-dimethylglutarate and 1 mM EDTA with an ioninestrength of 0.15 M adjusted by addition of NaCl, total volume 100 μL),the reaction mixture was monitored at UV absorbance at 254 nm andanalyzed by LC-MS at time points of 0 h, 3 h, 24 h and 48 h. LC-MSspectra at various time points showed no change in cefsulodin (retentiontime 6.1 min) and isonicotinamide (retention time 1.0 min, a marker ofcefsulodin degradation) concentrations, while the level of pNPP(retention time 2.0 and 3.5 min) decreased progressively with concurrentincrease of its hydrolyzed protein p-nitrophenol (retention time 8.6min), indicating SHP2 was active throughout the 48 hours. FIG. 16B:Incubation with cefsulodin at a higher concentration of SHP2 (10 μM)under the same conditions for 3 hours showed no cefsulodin degradation,indicating cefsulodin was not reacting with SHP2.

FIGS. 17A-17D depict QTOF-MS characterization of SHP2 inhibition bycefsulodin as analyzed in Example 9. FIG. 17A: ESI-MS analysis of SHP2indicated that it has a molecular weight of 32224.58. FIG. 17B: Afterincubation of cefsulodin (100 μM) and SHP2 (100 μM) in 50 mM3,3-dimethylglutarate and 1 mM EDTA with an ionic strength of 0.15 M(pH=7) for 3 hours, ESI-MS analysis showed only one peak at 32225.39.FIG. 17C: After incubation of cefsulodin (100 μM) and SHP2 (10 μM) in 50mM 3,3-dimethylglutarate and 1 mM EDTA with an ionic strength of 0.15 M(pH=7.0) for 3 hours, ESI-MS analysis again showed only one peak at32224.43. FIG. 17D: After incubation of phenyl vinyl sulfone (PVS) (100μM) and SHP2 (100 nM) in 50 mM 3,3-dimethylglutarate and 1 mM EDTA withan ionic strength of 0.15 M (pH 7) for 10 minutes, ESI-MS analysisshowed the unmodified SHP2 peak at 32224.20 and SHP2●PVS covalent adductat 32392.34. PVS has a molecular mass of 168.21.

FIGS. 18A-18F depict the co-crystal structure of SHP2 with alteredcefsulodin. FIG. 18A. Fo-Fc electron density map (contoured at 3.06)around the catalytic P-loop after the refinement of apo-form SHP2structure. FIG. 18B. 2Fo-Fc electron density map (contoured at 1.06)around the bound molecule (shown in stick) after the refinement of thecomplex structure. FIG. 18C. Chemical structure of the altered formobserved in co-crystal structure. The molecular weight of theunambiguously observed part is 430. FIG. 18D. The molecule (represent instick) binds into the active site of SHP2 (represent in surface) withabundant interactions with catalytic P-loop, pY-loop, Q-loop andWPD-loop. FIG. 18E. The detailed interactions between the alteredcefsulodin (stick, green carbon) and SHP2. Residues within 5 Å of thealtered cefsulodin are shown in white-carbon stick, and four loopsconstituting the active site pocket are shown in ribbon diagram andlabeled. FIG. 18F. Superimposing structures of PTP1B⋅phosphopeptidecomplex (PDBID: 1EEN) and SHP2⋅Cefsulodin reveals that SPAA overlapsvery well with pTyr residue in the phosphopeptide (cyan), suggesting itis a pTyr mimic

FIGS. 19A & 19B depict QTOF ESI-MS studies of re-dissolved crystals.FIG. 19A: The QTOF ESI-MS data of re-dissolved SHP2 crystals showssingle peak at 32224.57. FIG. 19B: The QTOF ESI-MS data of re-dissolvedco-crystal of SHP2 with Cefsulodin shows the protein peak at 32224.56and an additional peak at 32652.80, and the difference of 428.24corresponds to molecular mass of compound 1 after covalent bondformation between cefsulodin and SHP2.

FIGS. 20A-20C depict structure refinement, which shows the existence ofcovalent bond between compound 1 and C318 residue of SHP2. FIG. 20A: The2Fo-Fc electron density map (contoured at 1.06) around the backboneatoms (C, Cα and N, shown in stick) of the loop (residues 314-324) afterthe structure refinement. FIG. 20B: The compound 1 is covalently bondedto C318 of symmetric SHP2 molecule. FIG. 20C: The 2Fo-Fc electrondensity map (contoured at 1.06σ or 0.76σ) around the compound 1indicates the covalent bond formation.

FIGS. 21A-21E depict cefsulodin stability under various conditions asanalyzed in Example 9. FIG. 21A: LC-MS analysis of freshly preparedcefsulodin in pH 7.0 buffer containing 50 mM 3,3-dimethylglutarate and 1mM EDTA with an ionic strength of 0.15 M adjusted by addition of NaClshows cefsulodin (retention time 6.1 min) was very pure with tiny amountof nicotinamide (retention time 1.0 min). FIG. 21B: In MES buffer withpH=5.8, cefsulodin and isonicotinamide concentrations were almostunchanged at all time points of 12 h, 1 d, 2 d, indicating cefsulodinwas stable for at least 2 days. FIG. 21C: In CBTP buffer with pH=7.4,cefsulodin concentrations decreased progressively with the increase ofisonicotinamide concentration through time points of 12 h, 1 d, 2 d, theminor peak at retention time 7.9 min belongs to cefsulodin and CBTPconjugated product (see FIG. 21A). Minor peak (retention time 6.0 min)left to cefsulodin belongs to racemized cefsulodin. FIG. 21D: In CBTPbuffer with pH=9.1, cefsulodin concentrations decreased rapidly with theincrease of isonicotinamide concentration through time point of 3 h, 6h, 12 h, and cefsulodin was gone after 12 h. FIG. 21E: Incrystallization solution, cefsulodin concentrations decreasedprogressively with the increase of isonicotinamide concentration throughtime points of 3 h, 1 d, 2 d, and the majority of cefsulodin was goneafter 2 d, the minor peak at retention time 8.8 min belongs tocefsulodin and DTT conjugated product (see FIG. 21B).

FIGS. 22A-22D depict ESI-MS data of cefsulodin with buffer components.FIG. 22A: ESI-MS data of the minor peak at retention time 7.0 min inFIG. 22C middle spectrum shows the positive ion at 693.0 and thenegative ion at 691.0, indicating the existence of a molecule with massaround 692.0. FIG. 22B: ESI-MS data of the minor peak at retention time8.8 min in FIG. 21E middle spectrum shows the positive ion at 565.0 andthe negative ion at 563.0, indicating the existence of a molecule withmass around 564.0. FIG. 22C: The chemical structure, exact mass, andmolecule weight of a conjugate adduct of cefsulodin and CBTP,corresponding to the molecule with a retention time of 7.9 min in FIG.22C middle spectrum. FIG. 22D: The chemical structure, exact mass, andmolecule weight of a conjugate adduct of cefsulodin and DTT,corresponding to the molecule with a retention time of 8.8 min in FIG.22E middle spectrum.

FIGS. 23A & 23B depict the predicted binding modes of intact cefsulodinin complex with SHP2. FIG. 23A: Two probable binding modes with similarcalculated binding free energy. Cefsulodin is represented in stick, andSHP2 is represented in surface. FIG. 23B: Detailed interactions betweencefsulodin and SHP2in mode II. Residues within 5 Å distance of thecefsulodin are shown in white carbon stick, and four loops constitutingthe active site pocket are shown in ribbon diagram and highlighted.

FIGS. 24A-24C depict the design and synthesis of SPAA based novel SHP2inhibitors. FIG. 24A: The design of novel and stable SHP2 inhibitorsthat resemble cefsulodin structures. FIG. 24B: 4 sub-libraries weredesigned, in which carboxylic acid will couple with a set of 192 aminesto afford a total of 768 compounds. FIG. 24C: The chemical structures ofhits that inhibit SHP2 with good activity and specificity.

FIGS. 25A & 25B depict chemical structures of a set of 192 amines

FIGS. 26A-26E depict cellular activity of SPAA-based SHP2 inhibitors.FIG. 26A: MTT assay for compounds 2 to 7 in H1975 non-small cell lungcancer (NSCLC) cell line. Compounds 2, 5, 6 significantly (**p<0.01)reduced cell proliferation at 20 μM and 40 μM in a dose dependent mannerFIG. 26B: MTT assay of compounds 2 to 7 in MDA-MB-231 breast cancer cellline. Compounds 2, 5, 6 also significantly (**p<0.01) reduced cellsurvival at 20 μM and 40 μM in dose dependent manner FIG. 26C: In H1975lung cancer cells, compound 2 was able to decrease the EGF-mediatedERK1/2 phosphorylation in a dose dependent manner FIG. 26D: Thestructurally related negative control compound 7 failed to blockEGF-mediated ERK1/2 phosphorylation at 40 μM. FIG. 26E: compound 2 hadno effect on PMA-stimulated ERK1/2 phosphorylation.

FIGS. 27A & 27B depict the effect of compound 2 on inhibiting ERK1/2activation and growth of ErbB2+ breast cancer cells in a 3D Matrigelenvironment. FIG. 27A. SKBR3 cell growth in Matrigel over 5 days in thepresence of vehicle or the indicated concentrations of compound 2. FIG.27B. The levels of total and phospho ERK1/2 were detected by immunoblotfrom lysates prepared from cells recovered from Matrigel shown in FIG.27A.

FIG. 28 is a Western Blot showing the in vivo efficiency of L319N53 inblocking mPTPB activity as analyzed in Example 11.

FIGS. 29A-29C show the in vivo efficiency of L335N15 in sensitizing theinsulin signaling pathway in HepG2 cells as analyzed in Example 12.

DEFINITIONS

As intended herein, the terms “a” and “an” include singular as well asplural references unless the context clearly dictates otherwise. Forexample, the term “a PTP inhibitor” can include one or more suchinhibitors.

As used herein, “inhibition” or “inhibitory activity” each encompasswhole or partial reduction of activity or effect of an enzyme.

As used herein, “susceptible” and “at risk” refer to having littleresistance to a certain disease, disorder or condition, including beinggenetically predisposed, having a family history of, and/or havingsymptoms of the disease, disorder or condition.

“Treatment” as used herein includes the alleviation, prevention,reversal, amelioration or control of a pathology, disease, disorder,process, condition or event, such as diabetes, or the symptoms of suchpathology, disease, disorder, process, condition or event. In thiscontext, the term “treatment” is further to be understood as embracingthe use of a compound to inhibit, block, reverse, restrict or controlprogression of a disease, disorder, or condition associated withinappropriate activity of a protein phosphatase.

As used herein, the terms “pharmaceutical composition” and“pharmaceutical formulation” refer to compositions of matter comprisingat least one pharmaceutical compound.

The term “therapeutically suitable salt,” refers to salts or zwitterionsof pharmaceutical compounds which are water or oil-soluble ordispersible, suitable for treatment of disorders and effective for theirintended use. The salts may be prepared, for instance, during the finalisolation and purification of the compounds or separately by reacting anamino group of the compounds with a suitable acid. For example, acompound may be dissolved in a suitable solvent, such as, but notlimited to methanol and water, and treated with at least one equivalentof an acid, for instance hydrochloric acid. The resulting salt mayprecipitate out and be isolated by filtration and dried under reducedpressure. Alternatively, the solvent and excess acid may be removedunder reduced pressure to provide the salt. Representative salts includeacetate, adipate, alginate, citrate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate,digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate,formate, isethionate, fumarate, lactate, maleate, methanesulfonate,naphthylenesulfonate, nicotinate, oxalate, pamoate, pectinate,persulfate, 3-phenylpropionate, picrate, oxalate, maleate, pivalate,propionate, succinate, tartrate, trichloroacetate, trifluoroacetate,glutamate, para-toluenesulfonate, undecanoate, hydrochloric,hydrobromic, sulfuric, phosphoric, and the like. The amino groups of acompound may also be quaternized with alkyl chlorides, bromides, andiodides such as methyl, ethyl, propyl, isopropyl, butyl, lauryl,myristyl, stearyl, and the like.

Basic addition salts may be prepared, for instance, during the finalisolation and purification of pharmaceutical compounds by reaction of acarboxyl group with a suitable base such as the hydroxide, carbonate, orbicarbonate of a metal cation such as lithium, sodium, potassium,calcium, magnesium, or aluminum, or an organic primary, secondary, ortertiary amine. Quaternary amine salts may be derived, for example, frommethylamine, dimethylamine, trimethylamine, triethylamine, diethylamine,ethylamine, tributylamine, pyridine, N,N-dimethylaniline,N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine,dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine, andN,N′-dibenzylethylenediamine, ethylenediamine, ethanolamine,diethanolamine, piperidine, piperazine, and the like.

The term “therapeutically suitable prodrug,” refers to those prodrugs orzwitterions which are suitable for use in contact with the tissues ofsubjects and are effective for their intended use. The term “prodrug”refers to compounds that are transformed in vivo to a pharmaceuticalcompound, for example, by hydrolysis in blood. The term “prodrug,”refers to compounds that contain, but are not limited to, substituentsknown as “therapeutically suitable esters.” The term “therapeuticallysuitable ester,” refers to alkoxycarbonyl groups appended to the parentmolecule on an available carbon atom. More specifically, a“therapeutically suitable ester,” refers to alkoxycarbonyl groupsappended to the parent molecule on one or more available aryl,cycloalkyl and/or heterocycle groups. Compounds containingtherapeutically suitable esters are an example, but are not intended tolimit the scope of compounds considered to be prodrugs. Examples ofprodrug ester groups include pivaloyloxymethyl, acetoxymethyl,phthalidyl, indanyl and methoxymethyl, as well as other such groupsknown in the art.

The terms “specificity” or “selectivity” or “preference” of a certainPTP inhibitor for one PTP over another PTP, as described herein, isexpressed as fold of increase in inhibition activity, which iscalculated as the inverted ratio of IC₅₀ of the same inhibitor againstdifferent PTPs. For example, specificity for mPTPB over mPTPA equals[IC₅₀ (mPTPA)]/[IC₅₀ (mPTPB)].

The term “associated with abnormal activity of a protein tyrosinephosphatase” encompasses all diseases, disorders, or conditions in whichsymptoms are, in part, related to excessive or deficient activity of aprotein tyrosine phosphatase, as compared to the activity of the proteintyrosine phosphatase of a subject without such diseases, disorders, orconditions.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure is generally directed to small moleculeinhibitors of PTPs and their uses as effective therapeutics, and moreparticularly, to inhibitors of mPTPA, mPTPB, LMWPTP, Laforin, SHP2, LYP,and HePTP. These inhibitors can be administered totreat/control/mitigate diseases and conditions including cancer,diabetes, infectious and neurological diseases.

Generally, the present disclosure provides a library of compoundsderived from α-sulfophenylacetic amide (SPAA) as novel sulfonic acidbased pTyr mimetics. These compounds have a structure of Formula 1:

or a therapeutically suitable prodrug thereof or a therapeuticallysuitable salt thereof, wherein R₁ is hydrogen and R₂ is selected fromC₁-C₁₀ alkyl, aryl, heteroaryl, —NH—R_(2a), —(CH₂)_(m)NH—CO—R_(x), and—(CH₂)_(n)—R_(2b)—(CH₂)_(q)—NH—CO—CO—NH—R_(y);

-   -   wherein, when R₂ is aryl or heteroaryl, R₂ is optionally        substituted with one or more substituent selected from the group        consisting of C₁-C₄ alkyl, C₁-C₄ alkoxy, C₁-C₄ alkoxy carbonyl,        amino, aryl, benzyloxy (—OBn), —CF₃, carboxy, halogen,        1-imidazolyl, 4-morpholinyl, nitro, and —(CH₂)_(s)—NH—CO—R_(z);    -   wherein m, n, q, and s independently are 0-4;    -   wherein R_(2a) and R_(2b) independently are aryl;    -   wherein R_(x), R_(y), and R_(z) independently are aryl or        heteroaryl, and the aryl or heteroaryl are independently        optionally substituted with one or more substituent selected        from the group consisting of C₁-C₄ alkyl, benzoyl, benzyl,        benzyloxy (—OBn), phenyl, halogen, 1H-benzimidazole-2-yl, and        2-thiophenyl;

-   or wherein R₁, R₂, and the N atom to which they are attached are    joined together to form a monocyclic or bicyclic heterocycle;

-   wherein R₃ is hydrogen or halogen; and

-   wherein R₄ is hydrogen or aryl, the aryl being optionally    substituted with one or more substituent selected from the group    consisting of halogen, C₁-C₄ alkyl, C₁-C₄ alkoxy, phenyl, nitro,    cyano, and —COCF₃.

Based on the structure of α-sulfophenylacetic amide (SPAA), threelibraries of compounds are provided herein. A total of more than 5000compounds have been synthesized through peptide coupling reaction using192 carboxylic acids (for free amines intermediates) and 192 amines (forfree carboxylic acid intermediates). As shown in FIGS. 3A-3C, the threelibraries of compounds are derived from 3 groups of SPAA analogs: GroupA with diamine linker, Group B with substitution on the phenyl residueor on the α-carbon, and Group C with oxalyl linkers. Coupling of thelibrary of carboxylic acids or amines with the corresponding amino orcarboxy SPAA analogs in Groups A-C through peptide bond formationprovides exemplary compounds of Formula 1 (typically with a molecularweight (MW) of 200 to 700).

Accordingly, in Formula 1, R₁ and R₂ can be separate moieties and R₁ ishydrogen. In some embodiments, R₁ is hydrogen, and R₂ is aryl orheteroaryl. Suitably, in some embodiments, R₂ is phenyl orbenzo[d]thiazol-2-yl, optionally substituted with one or moresubstituent selected from the group consisting of C₁-C₄ alkyl, C₁-C₄alkoxy, C₁-C₄ alkoxy carbonyl, amino, aryl, benzyloxy (—OBn), —CF₃,carboxy, halogen, 1-imidazolyl, 4-morpholinyl, and nitro.

More particularly, in one aspect for example, the present disclosureprovides compounds of Formula 1a:

or a therapeutically suitable prodrug thereof or a therapeuticallysuitable salt thereof, wherein R₁ is hydrogen and R₂ is selected fromC₁-C₁₀ alkyl, aryl, heteroaryl, —NH—R_(2a), —(CH₂)_(m)NH—CO—R_(x), and—(CH₂)_(n)—R_(2b)—(CH₂)_(q)—NH—CO—CO—NH—R_(y);

-   -   wherein, when R₂ is aryl or heteroaryl, R₂ is optionally        substituted with one or more substituent selected from the group        consisting of C₁-C₄ alkyl, C₁-C₄ alkoxy, C₁-C₄ alkoxy carbonyl,        amino, aryl, benzyloxy (—OBn), —CF₃, carboxy, halogen,        1-imidazolyl, 4-morpholinyl, and nitro;    -   wherein m, n, and q independently are 0-4;    -   wherein R_(2a) and R_(2b) independently are aryl;    -   wherein R_(x) and R_(y) independently are aryl or heteroaryl,        and the aryl or heteroaryl are independently optionally        substituted with one or more substituent selected from the group        consisting of C₁-C₄ alkyl, benzoyl, benzyl, benzyloxy (—OBn),        phenyl, halogen, 1H-benzimidazole-2-yl, and 2-thiophenyl;

-   or wherein R₁, R₂, and the N atom to which they are attached are    joined together to form a monocyclic or bicyclic heterocycle;

-   wherein R₃ is hydrogen or halogen; and

-   wherein R₄ is hydrogen or aryl, the aryl being optionally    substituted with one or more substituent selected from the group    consisting of halogen, C₁-C₄ alkyl, C₁-C₄ alkoxy, phenyl, nitro,    cyano, and —COCF₃.

In another aspect for example, the present disclosure provides compoundsof Formula 1b:

or a therapeutically suitable prodrug thereof or a therapeuticallysuitable salt thereof, wherein R1 is hydrogen and R2 is aryl orheteroaryl and is substituted with —(CH₂)_(s)—NH—CO—R_(z);

-   -   wherein s is 0-4;    -   wherein R_(z) is aryl or heteroaryl, and the aryl or heteroaryl        are independently optionally substituted with one or more        substituent selected from the group consisting of C₁-C₄ alkyl,        benzoyl, benzyl, benzyloxy (—OBn), phenyl, halogen,        1H-benzimidazole-2-yl, and 2-thiophenyl;

-   or wherein R₁, R₂, and the N atom to which they are attached are    joined together to form a monocyclic or bicyclic heterocycle;

-   wherein R₃ is hydrogen or halogen; and

-   wherein R₄ is hydrogen or aryl, the aryl being optionally    substituted with one or more substituent selected from the group    consisting of halogen, C₁-C₄ alkyl, C₁-C₄ alkoxy, phenyl, nitro,    cyano, and —COCF₃.

By way of further examples, as shown in Table 1, R₂ can be a phenylsubstituted with bromo and methyl groups (L319M34), or R₂ can be abenzo[d]thiazol-2-yl moiety (L319-15). In other embodiments, the —NH—R₂structure is derived from a diamine linker (i.e. library of Group Acompounds), such as the ones shown in L319-02-A60 and L319-05-A64 (Table2). In other embodiments, the —NH—R₂ structure is derived from an oxalyllinker, which is coupled to the α-sulfophenylacetyl moiety through adiamine linker, such as the ones shown in L319-21-M52 (Table 2),L319-20-M24 (Table 3), and L319-24-M77 (Table 3).

TABLE 1

L319M34

L319M52

L319M54

L319N15

L319N22

L319N46

L319N47

L319N53

TABLE 2

L319M50

L319M52

L319M63

L319M73-2

L319-02-A60

L319N08

L319N72

L319M78

L319-05-A64

L319-21-M06

L319-21-M50

L319-21-M51

L319-21-M52

TABLE 3

L319-20-M24

L319-20-M74

L319-20-M78

L319-20-N08

L319-20-N25

L319-22-M34

L319-22-M50

L319-22-M51

L319-23-M74

L319-24-M74

L319-24-M77

L319-04-B34

In other embodiments, compounds in which the α-phenyl is replaced by ahydrogen atom are also provided, such as L335N15-07 and L335M34 shown inTable 4. With respect to R₃ and R₄ of Formula 1, as shown in FIG. 3B,the hydrogen on the α-carbon of the α-sulfophenylacetyl moiety can bereplaced by halogen (such as bromo), and the phenyl group can bereplaced by other aromatic groups (such as naphthalenyl). In addition,the aromatic group on the α-carbon of the α-sulfophenylacetyl moiety canhave substituents such as halogen, C₁-C₄ alkyl, phenyl, nitro, andCOCF₃.

TABLE 4

L319M34

L319M54

L319M68

L319M88

L319N15

L319N22

L335N15

L335N15-07

L335M34

L335M54

L335M68

L335M88

In yet another embodiment, R₁, R₂, and the N atom to which they areattached are joined together to form a monocyclic or bicyclicheterocycle, such as those shown in L319N46, L319N47, and L319N53 (Table1). Preferably, R₁, R₂, and the N atom form a heterocycle selected fromthe group consisting of:

The present disclosure further provides a compound of Formula 2:

-   wherein R₂′ is heterocycle, optionally substituted with one or more    substituent selected from the group consisting of C₁-C₄ alkyl, C₁-C₄    alkoxy, C₁-C₄ alkoxy carbonyl, amino, aryl, benzyloxy (—OBn), —CF₃,    carboxy, halogen, 1-imidazolyl, 4-morpholinyl, and nitro;-   wherein R₃′ is hydrogen or halogen; and-   wherein R₄′ is hydrogen or aryl, the aryl being optionally    substituted with one or more substituent selected from the group    consisting of halogen, C₁-C₄ alkyl, C₁-C₄ alkoxy, phenyl, nitro,    cyano, and —COCF₃.

Other non-limiting examples of the novel α-sulfophenylacetic amidecompounds are included in the present disclosure, as shown in theExamples and Tables 1-7.

TABLE 5

L319M34

L319M50

L319M54

L319M63

L319M68

L319M88

L319N15

L319N22

TABLE 6

L319N13

L319N54

L319N15-01

L319N15-02

L319N15-03

L319N15-04

L319N15-05

L319N15-06

L319N15-07

L319N15-08

L319N15-09

L3169N15-10

L319N15-11

L319N15-12

L319N15-13

L319N15-14

L319N15-15

L319N15-16

L319N15-17

L319N15-18

L338N15

L335N15

L335N15-07

TABLE 7

L319-06-M68

L319-07-M68

L319-08-N58

L319-11-M68

L319-12-M68

L319-13-M68

L319-14-M50

L319-14-M68

L319-14-N03

L319-16-M47

L319-16-M60

L319-16-M93

L319-16-N55

L319-16-N58

L319-14

L319-14

L319-16

L319-Br1-N15

L319-Br1-N47

L319-Br1-N76

The present disclosure further provides a compound of Formula 3:

or a therapeutically suitable prodrug thereof or a therapeuticallysuitable salt thereof, wherein R is aryl or heteroaryl, optionallysubstituted with one or more substituent selected from the groupconsisting of C₁-C₄ alkyl, halogen, 1-imidazolyl, benzyl, and2-thiophenyl. These compounds have been found to specifically inhibitSHP2 and can be administered to treat/control/mitigate diseases andconditions including cancer, diabetes, infectious and neurologicaldiseases. Particularly, these inhibitors can be administered totreat/control/mitigate cancers such as breast cancer, lung cancer, coloncancer, prostate cancer, neuroblastoma, glioblastoma, melanoma,hepatocellular carcinoma, and leukemia.

Particularly suitable compounds of Formula 3 are shown in Table 8 below.

TABLE 8 Compound Structure 2

3

4

5

6

7

As shown in the Examples, the SPAA based compounds of the presentdisclosure are effective inhibitors of various PTPs. In someembodiments, the compounds disclosed herein can be used to inhibitmPTPA, mPTPB, LMWPTP, and Laforin with unprecedented potency andspecificity. These PTPs are targets for the treatment of diseasesassociated with abnormal protein tyrosine phosphatase activity (such astuberculosis, cancer, Lafora disease, and type 2 diabetes). Accordingly,the present disclosure also provides the use of the compounds of Formula1, a therapeutically suitable prodrug thereof, or a therapeuticallysuitable salt thereof, as well as the compounds of Formula 2 disclosedherein as PTP inhibitors for treating these diseases.

In one aspect, the present disclosure provides a method of inhibiting aprotein tyrosine phosphatase (PTP) selected from the group consisting ofmPTPA, mPTPB, low molecular weight PTP (LMWPTP), and Laforin in asubject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of the compound of Formula1a, a therapeutically suitable prodrug thereof, or a therapeuticallysuitable salt thereof, or the compound of Formula 2.

In another aspect, the present disclosure provides a method of treatingtuberculosis in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of thecompound of Formula 1a, a therapeutically suitable prodrug thereof, or atherapeutically suitable salt thereof, or the compound of Formula 2.

In another aspect, the present disclosure provides a method of treatinga cancer in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of thecompound of Formula 1a, a therapeutically suitable prodrug thereof, or atherapeutically suitable salt thereof, or the compound of Formula 2. Insome embodiments, the cancer can be one selected from the groupconsisting of breast cancer, colon cancer, bladder cancer, and kidneycancer.

In another aspect, the present disclosure provides a method of treatingLafora disease in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of thecompound of Formula 1a, a therapeutically suitable prodrug thereof, or atherapeutically suitable salt thereof, or the compound of Formula 2.

In another aspect, the present disclosure provides a method of treatingtype 2 diabetes in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of thecompound of Formula 1a, a therapeutically suitable prodrug thereof, or atherapeutically suitable salt thereof, or the compound of Formula 2.

In another aspect, the present disclosure provides a method ofinhibiting SHP2 in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of thecompound of Formula 3, a therapeutically suitable prodrug thereof, or atherapeutically suitable salt thereof.

In another aspect, the present disclosure provides a method of treatingcancer in a subject in need thereof, the method comprising administeringto the subject a therapeutically effective amount of the compound ofFormula 3, a therapeutically suitable prodrug thereof, or atherapeutically suitable salt thereof. In some embodiments, the cancercan be one selected from the group consisting of breast cancer, lungcancer, colon cancer, prostate cancer, neuroblastoma, glioblastoma,melanoma, hepatocellular carcinoma, and leukemia.

Some subjects that are in specific need of treatment for disease andconditions as discussed above (e.g., tuberculosis, cancer, Laforadisease, type 2 diabetes) may include subjects who are susceptible to,or at elevated risk of, experiencing tuberculosis, cancer (e.g., breastcancer, lung cancer, colon cancer, prostate cancer, neuroblastoma,glioblastoma, melanoma, hepatocellular carcinoma, and leukemia), Laforadisease, type 2 diabetes, and the like. Subjects may be susceptible to,or at elevated risk of, experiencing tuberculosis, cancer, Laforadisease, type 2 diabetes due to family history, age, environment, and/orlifestyle. Based on the foregoing, because some of the methodembodiments of the present disclosure are directed to specific subsetsor subclasses of identified subjects (that is, the subset or subclass ofsubjects “in need” of assistance in addressing one or more specificconditions noted herein), not all subjects will fall within the subsetor subclass of subjects as described herein for certain diseases,disorders or conditions.

In another aspect, the present disclosure provides a pharmaceuticalcomposition comprising a compound of Formula 1a, a therapeuticallysuitable prodrug thereof, or a therapeutically suitable salt thereof, orthe compound of Formula 2 and a pharmaceutically acceptable carrier.

In another aspect, the present disclosure provides a pharmaceuticalcomposition comprising a compound of Formula 3, a therapeuticallysuitable prodrug thereof, or a therapeutically suitable salt thereof,and a pharmaceutically acceptable carrier.

Representative pharmaceutical formulations include those suitable fororal, parenteral (including subcutaneous, intradermal, intramuscular andintravenous) and rectal administration. The formulations may bepresented in unit dosage form and may be prepared by any of the methodswell known in the art of pharmacy. Usually, the formulations areprepared by uniformly and intimately bringing into association theactive ingredient with a liquid carrier, or a finely divided solidcarrier, or both, and then, if necessary, forming the associated mixtureinto the desired formulation.

As used herein, the phrase “pharmaceutically acceptable” refers to thoseligands, materials, formulations, and/or dosage forms which are, withinthe scope of sound medical judgment, suitable for use in contact withthe tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. The phrase“pharmaceutically acceptable carrier”, as used herein, refers to apharmaceutically acceptable material, formulation or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the active compound fromone organ or portion of the body, to another organ or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other components of the formulation and not injurious to thesubject. Lyophilized formulations, which may be reconstituted andadministered, are also within the scope of the present disclosure.

Pharmaceutical formulations suitable for oral administration may bepresented as discrete units, such as a capsule, cachet, tablet, orlozenge, each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or a suspension in anaqueous liquid or a non-aqueous liquid such as a syrup, elixir or adraught, or as an oil-in-water liquid emulsion or a water-in-oil liquidemulsion. The formulation may also be a bolus, electuary or paste.

A tablet may be made by compressing or molding a pharmaceutical compoundwith the pharmaceutically acceptable carrier. Compressed tablets may beprepared by compressing in a suitable machine the active ingredient in afree-flowing form, such as a powder or granules, in admixture with, forexample, a binding agent, an inert diluent, a lubricating agent, adisintegrating agent and/or a surface active agent. Molded tablets maybe prepared by molding in a suitable machine a mixture of the powderedactive ingredient moistened with an inert liquid diluent. The tabletsmay optionally be coated or scored and may be formulated so as toprovide slow or controlled release of the active ingredient.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions, and may also include anantioxidant, buffer, a bacteriostat and a solution which renders thecomposition isotonic with the blood of the recipient, and aqueous andnon-aqueous sterile suspensions which may contain, for example, asuspending agent and a thickening agent. The formulations may bepresented in single unit-dose or multi-dose containers, and may bestored in a lyophilized condition requiring the addition of a sterileliquid carrier prior to use.

The compounds are administered in a therapeutically effective amount toprovide treatments of the above-described diseases and disorders. Thephrase “therapeutically effective amount” of the compound of thedisclosure means a sufficient amount of the compound to treat disorders,at a reasonable benefit/risk ratio applicable to any medical treatment.It can be understood, however, that the total daily usage of thecompounds of the disclosure can be decided by the attending physicianwithin the scope of sound medical judgment. The specific therapeuticallyeffective dose level for any particular patient can depend upon avariety of factors including the disorder being treated and the severityof the disorder; activity of the specific compound employed; thespecific pharmaceutical composition employed; the age, body weight,general health, sex and diet of the patient; the time of administration,route of administration, and rate of excretion of the specific compoundemployed; the duration of the treatment; drugs used in combination orcoincidental with the specific compound employed; and like factorswell-known in the medical arts. For example, it is well within the skillof the art to start doses of the compound at levels lower than requiredto achieve the desired therapeutic effect and to gradually increase thedosage until the desired effect is achieved.

Actual dosage levels of compounds in the pharmaceutical compositions ofthis disclosure can be varied so as to obtain an amount of thecompound(s) that is effective to achieve the desired therapeuticresponse for a particular patient, compositions and mode ofadministration. The selected dosage level can depend upon the activityof the particular compound, the route of administration, the severity ofthe condition being treated and the condition and prior medical historyof the patient being treated. However, it is within the skill of the artto start doses of the compound at levels lower than required to achievethe desired therapeutic effect and to gradually increase the dosageuntil the desired effect is achieved.

This written description uses examples to disclose the invention,including the best mode, and also to enable any person skilled in theart to practice the invention, including making and using any devices orsystems and performing any incorporated methods. The patentable scope ofthe invention is defined by the claims, and may include other examplesthat occur to those skilled in the art. Such other examples are intendedto be within the scope of the claims if they have structural elementsthat do not differ from the literal language of the claims, or if theyinclude equivalent structural elements with insubstantial differencesfrom the literal languages of the claims.

EXAMPLES

Materials and general procedures. p-Nitrophenyl phosphate (pNPP) waspurchased from Fluke Co. Dithiothreitol (DTT) was provided by Fisher(Fair Lawn, N.J.). For organic synthesis, reagents were used aspurchased (Aldrich, Acros, Alfa Aesar, TCI) except where noted. ¹H and¹³C NMR spectra were obtained on a BrukerAvance II 500 MHz NMRspectrometer with TMS or residual solvent as standard. Mass spectra wereobtained using an Agilent Technologies 6130 quadrupole LC/MS. HPLCpurification was carried out on a Waters Delta 600 equipped with aSunfire Prep C18 OBD column (30 mm, 150 mm, 5 μm) with methanol-water(both containing 0.1% TFA) as mobile phase (gradient: 50-100% methanol,flow 10 mL/min). The purity of all final tested compounds wasestablished to be >95% Agilent Technologies 6130 quadrupole LC/MS byusing methanol-water (both containing 0.1% TFA) as the mobile phase(gradient: 30-100% methanol, flow 1.0 mL/min), with UV monitoring at thefixed wavelength of 254 nm.

Representative procedure for the synthesis of products. To α-Sulfoα-phenyl acetyl chloride (0.234 g, 1 mmol) and DIEA (0.522 mL, 3 mmol)in DMF (2 mL) was added propyl amine (0.09 mL, 1.1 mmol), and themixture was stirred at room temperature for 1 hour. After quenching withwater, it was subjected to HPLC purification, and product 5 (L319N01)was obtained as colorless oil (93% yield, >95% purity). ¹H NMR (500 MHz,CDCl3) δ 8.23 (s, 1H), 7.45-7.44 (m, 2H), 7.27-7.20 (m, 3H), 4.42 (s,1H), 3.11-3.02 (m, 2 H), 1.46-1.39 (m, 2H), 0.85 (t, J=7.4 Hz, 3H); ¹³CNMR (500 MHz, CDCl3) δ 167.3, 135.7, 129.6, 127.4, 126.8, 71.5, 40.4,22.3, 11.4. ESI-MS cacld. for C₁₁H₁₆NO₄S (M+H⁺): m/z 258.1; found 258.0.

Library synthesis in 96-well plate. To each well of a 96-well plate wasadded 4 (20 μL, 20 mM in DMF), HBTU (20 μL, 20 mM in DMF), HOBt (20 μL,20 mM in DMF) and DIEA (20 μL, 75 mM in DMF). Five minutes later,various amines (1 μL, 500 mM in DMF) were added. The plate was allowedto stand at room temperature overnight to give products in stocksolutions at 4 mM (assuming the product is obtained at 80% yield).

Protein expression and purification; Kinetic parameters determination;and inhibition study. These procedures are carried out according topreviously reported methods (Combs, J. Med. Chem. 2010, 53, 2333-2344;Thareja et al., Med. Res. Rev. 2011, early view).

Expression and Purification of Recombinant mPTPB:

pET28b-mPTPB (from Dr. Christoph Grunder, University of California,Berkeley) was used to transform into E. coli BL21/DE3 and grown in LBmedium containing 50 μg/ml kanamycin at 37° C. to an OD600 of 0.5.Following the addition of IPTG to a final concentration of 20 μM, theculture was incubated at 20° C. with shaking for an additional 16 hours.The cells were harvested by centrifugation at 5000 rpm for 5 minutes at4° C. The bacterial cell pellets were resuspended in 20 mM Tris, pH 7.9,500 mM NaCl, 5 mM imidazole, and were lysed by passage through a Frenchpress cell at 1,200 p.s.i. twice. Cellular debris was removed bycentrifugation at 16,000 rpm for 30 minutes at 4° C. The protein waspurified from the supernatant using standard procedures ofNi-nitrilotriacetic acid-agarose (Qiagen) affinity purification. Theprotein eluted from Ni-NTA column was concentrated with an Amicon Ultracentrifugal filter device (Millipore), and the buffer was changed to 20mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA and 1 mM DTT. Proteinconcentration was determined using the Bradford dye binding assay(Bio-Rad) diluted according to the manufacturer's recommendations withbovine serum albumin as standard. The purified mPTPB were made to 20%glycerol and stored at −20° C.

Kinetic Characterization of mPTPB Inhibitors:

The inhibition assays were performed at 25° C. in 50 mM3,3-dimethylglutarate buffer, pH 7.0, containing 1 mM EDTA with an ionicstrength of 0.15M adjusted by NaCl. The salicylic acid based library wasscreened in a 96-well format at 1 μM compound concentration. Thereaction was started by the addition of 5 μl of the enzyme to 195 μl ofreaction mixture containing 2.5 mM (the Km value) of pNPP and variousconcentrations of the inhibitor. The reaction was quenched after 5minutes by the addition of 50 μl of 5N NaOH, and then 200 μl of reactionmixture was transferred to a 96-well plate. The absorbance at 405 nm wasdetected by a Spectra MAX340 microplate spectrophotometer (MolecularDevices). IC₅₀ values were calculated by fitting the absorbance at 405nm versus inhibitor concentration to the following equation:

A ₁ /A ₀ =IC ₅₀/(IC ₅₀ +[I])

where A₁ is the absorbance at 405 nm of the sample in the presence ofinhibitor; A₀ is the absorbance at 405 nm in the absence of inhibitor;and [I] is the concentration of the inhibitor.

The inhibition constants (Ki) for the inhibitor for mPTPB weredetermined at pH 7.0 and 25° C. The mode of inhibition and Ki value weredetermined in the following manner: at various fixed concentrations ofinhibitor (0-3 Ki), the initial rate at a series of pNPP concentrationswas measured by following the production of p-nitrophenol as describeabove, ranging from 0.2- to 5-fold the apparent Km values. The data werefitted SI-23 to appropriate equations using SigmaPlot-Enzyme Kinetics toobtain the inhibition constant and to assess the mode of inhibition. Forselectivity studies, the PTPs, including mPTPA, YopH, CD45, FAP-1,HePTP, Lyp, PTP1B, SHP1, SHP2, and VHX were expressed and purified fromE. coli. The inhibition assay for these PTPs were performed under thesame conditions as mPTPB except using a different pNPP concentrationcorresponding to the K_(m) of the PTP studied.

Cellular Studies:

Raw264.7 mouse macrophages were cultured in Dulbecco's modified Eagle'smedium (DMEM) supplemented with 10% FBS (Invitrogen), penicillin (50units/mL), and streptomycin (50 μg/mL) under a humidified atmospherecontaining 5% CO₂ at 37° C. Transfected Raw264.7 cells (Vector,WT-mPTPB) were seeded in a 12-well plate at a density of 4×10⁴cells/well. The following day cells were treated with mPTPB inhibitor11a for 1 hour, then stimulated with IFN-γ (200 U/ml) for 1 hour.Subsequently, the cells were washed with ice-cold phosphate bufferedsaline, and lysed with lysis buffer on ice for 30 minutes. Cell lysatewas then cleared by centrifuging at 13,000 rpm for 15 minutes. Thephosphorylation of ERK1/2 was detected by Western blotting.

Example 1

Construction of SPAA based focused library. Previously, cefsulodin (aβ-lactam antibiotics, structure shown in FIG. 2) was identified as aninhibitor of mPTPB. The crystal structure of SHP2-cefsulodin complexshows that sulfonic acid is in close proximity with the PTP signaturemotif, the α-phenyl group forms a π-π interaction with P loop residuePhe, indicating the nature of SPAA as pTyr mimetic (FIG. 2). Withoutbeing bound by any theory, it is believed that SPAA is completelydifferent from conventional pTyr mimetics (such as F2PMP, salicylicacid, etc.), in which the acid group and additional fragment are locatedon opposite sides of the benzene ring (FIG. 1). For SPAA, however, theadditional fragment needs to be extended from the same side of thebenzene ring as the acid group. This would have been considered as adisruption to the binding due to the valley shape of the PTP activesite, thus would have been avoided during the design of a conventionalPTP inhibitor.

Surprisingly, it has been found that the amide group in cefsulodin doesnot disrupt the binding of sulfonic acid and PTP signature motif butinstead extends out from the pocket and forms hydrogen bonds with activesite entrance residues. In addition, SPAA, the sulfonic acid based pTyrmimetic, has been shown to have high affinity to mPTPB and LMWPTP(IC₅₀=200 μM, and 2 mM, respectively), good water solubility (>100mg/mL), and it is commercially available and inexpensive.

To demonstrate the utility of these novel pTyr mimetics as potent andselective PTP inhibitors, 3 libraries of compounds were designed to linkSPAA with either a set of carboxylic acids or amines (FIGS. 3A-3C). Thesynthesis processes of the precursors for each library are shown inFIGS. 4-6, respectively.

Amide/peptide bond formation is an efficient and reliable method forlibrary constructions; and it allows the use of the most common andcommercially available amines and carboxylic acids as reactants. Inpractice, all carboxylic acid and amine building blocks ofSigma-Aldrich, and selected representative 192 carboxylic acids and 192amines with low molecular weight covering almost all the chemo typesfrom their collections were examined. The library was assembled directlyon 96-well plates by standard HBTU peptide coupling conditions. Thewells from each plate were monitored by LC-MS, which indicated theproduction of various compounds from the reactions. Thus, a total of5184 pTyr mimetic compounds were obtained with molecular weights from200 to 700. These compounds were immediately subjected to screeningagainst a panel of PTP enzymes, including cytosolic PTPs, PTP1B, TC-PTP,SHP2, LYP, HePTP, and FAP1, the receptor-like PTPs, CD45, LAR, and PTPα,the dual specificity phosphatases MKP3, VHR, VHX, CDC14A, CDC25A,LMWPTP, bacterial PTPs mPTPA, and mPTPB, and SSU72. The screeningconcentrations were initially set at 10 μM for each PTP, and werereduced to 1 μM and 0.1 μM if the inhibition scores were high forcertain PTPs. Top and selective hits were resynthesized by this methodand purified by reversed phase HPLC to >95% pure. The IC₅₀ of thesecompounds against a panel of PTPs were measured, as is summarized inTable 9.

TABLE 9 IC₅₀ values of mPTPB inhibitors against a panel of PTPs IC₅₀(μM) L319M34 L319M52 L319M54 L319N15 L319N22 L319N46 L319N47 L319N53mPTPB 0.055 0.106 0.078 0.116 0.203 0.060 0.009 0.020 mPTPA 0.196 0.560.265 0.194 0.972 >200 11.3 >200 LMWPTP 5.29 72.27 7.96 7.33 6.39 >200115 >200 Laforin 4.05 5.08 4.25 2.1 27 >200 61 >200YopH >200 >200 >200 >200 >200 >200 160 >200 TbPTP1 >200118 >200 >200 >200 >200 38 >200 PTP1B >200 >200 >200 >200 >200 >200145 >200 TcPTP >200 170 >200 >200 >200 >200 160 >200LYP >200 >200 >200 >200 >200 >200 42 >200 PEST 72.3 77 >200 43.5 67 >20042 >200 SHP1 >200 >200 >200 >200 >200 >200 66 >200SHP2 >200 >200 >200 >200 >200 >200 76 >200 HePTP >200 130 >200 5094 >200 77 >200 Meg2 >200 >200 >200 >200 >200 >200 147 >200PTPH1 >200 >200 >200 >200 >200 >200 30 >200 FAP1 >200 88 180 34 76 >20021 76 CD45 >200 >200 >200 >200 >200 >200 140 >200LAR >200 >200 >200 >200 >200 >200 >200 >200PTPα >200 >200 >200 >200 >200 >200 >200 >200PTPβ >200 >200 >200 >200 >200 >200 50 >200PTPγ >200 >200 >200 >200 >200 >200 180 >200PTPσ >200 >200 >200 >200 >200 >200 >200 >200PTPε >200 >200 >200 >200 >200 >200 >200 >200PTPμ >200 >200 >200 >200 >200 >200 >200 >200 STEP >200 >200 >200 >200140 >200 >200 >200 MKP3 >200 >200 >200 >200 >200 >200 >200 >200VHR >200 >200 >200 >200 >200 >200 99 >200 VHX 92 170 >200 >200 >200 >200150 >200 VHZ >200 >200 >200 >200 >200 >200 80 >200CDC14A >200 >200 >200 >200 >200 >200 >200 >200PP5 >200 >200 >200 >200 >200 >200 >200 >200 Ssu72 >200 97.6 >200 6983 >200 >200 >200

Example 2

Potent and specific inhibition of mPTPB. SPAA based compounds from thelibraries were studied as inhibitors against mPTPB, a virulence factorof Mtb strain and a novel drug target of tuberculosis (TB).

Compound L319N47 demonstrated an IC₅₀ of 9 nM against mPTPB, a 2000-foldincrease in potency compared to SPAA, (Tables 1 and 9). L319N47 alsodemonstrated a 1000-fold preference for mPTPB over mPTPA (IC₅₀=11.3 μM)and a greater than 2000-fold preference for mPTPB over 30 other PTPs(e.g. LMWPTP, IC₅₀=115 μM), including cytosolic PTPs, PTP1B, TC-PTP,SHP2, Lyp and FAP1, the receptor-like PTPs, CD45, LAR, and PTPα, thedual specificity phosphatases VHR, VHX, CDC14A, and the LMWPTP.

L319N46 and L319N53 (IC₅₀=60 and 20 nM, respectively) were slightly lessactive than L319N47. However, they demonstrated even higher specificitytowards mPTPB. For example, L319N53 exhibited a greater than 10000-foldselectivity over any PTP tested. It is believed that any previouslyreported inhibitors of PTP, including mPTPB, possesses no more than100-fold specificity (see, for example, Noren-Muller et al., Proc. Natl.Acad. Sci. USA 2006, 103, 10606-10611; Correa et al., Chem. Asian J.2007, 2, 1109-1126; Noren-Muller et al., Angew. Chem. Int. Ed. 2008, 47,5973-5977; Weide et al., Bioorg. Med. Chem. Lett. 2006, 16, 59-63;Soellner et al., J. Am. Chem. Soc. 2007, 129, 9613-9615; Grundner etal., Structure 2007, 15, 499-509; Tan et al., Org. Lett. 2009, 11,5102-5105; Chen et al., ACS Med. Chem. Lett. 2010, 1, 355-359; Vintonyaket al., Angew. Chem. Int. Ed. 2010, 49, 5902-5905), suggesting that thepotency and specificity of the compounds described herein areunprecedented.

Remarkably, L319N46, L319N47, and L319N53 share a common heterocyclicstructure, which includes the nitrogen atom in the amide group, adjacentto the pTyr mimetic (i.e. the Ph—CH(SO₃H)— group). For L319N46 andL319N47, the heterocyclic structure comprises a piperidine moiety; forL319N53, the heterocyclic structure comprises a decahydroquinolinemoiety (Table 1). Without being bound by any theory, it is hypothesizedthat a structure-activity relationship underlies the exceptional potencyand specificity of these three compounds. It is further hypothesizedthat the compact structures, low molecular weights (MW about 400), andwater solubility (>10 mg/mL) are among the factors that contribute totheir drug-likeness. These results indicate that SPAA-based compoundsL319N46, L319N47, and L319N53 are currently the most potent and specificinhibitors of mPTPB, allowing for the development of new tuberculosis(TB) agents targeting mPTPB.

Example 3

General Procedures for the Preparation of Inhibitors. Reagents were usedas purchased from Sigma-Aldrich and Fisher Scientific. ¹H and ¹³C NMRspectra were obtained on a BrukerAvance II 500 MHz NMR spectrometer withtetrame-thylsilane or residual solvent as standard. Mass spectra wereobtained using an Agilent Technologies 6130 quadrupole LC/MS. HPLCpurification was carried out on a Waters Delta 600 equipped with aSunfire Prep C18 OBD column (30 mm/150 mm, 5 μm) with methanol-water(both containing 0.1% TFA) as the mobile phase (gradient: 50-100%methanol, flow 10 mL/min). The purity of all final tested compounds wasestablished to be >95% by Agilent Technologies 6130 quadrupole LC/MS byusing methanol-water (both containing 0.1% TFA) as the mobile phase(gradient: 0-100% methanol, flow 1.0 mL/min), with UV monitoring at thefixed wavelength of 254 nm.

Synthesis of L335-M34 (mPTPA). To a round-bottom flask were addedsulfoacetic acid (0.14 g, 1.0 mmol), DMF (5 mL), HBTU (0.379 g, 1 mmol),3,5-dibromo-4-methylaniline (0.265 g, 1 mmol), DIEA (0.52 mL, 3 mmol),and DMAP (0.012 g, 0.1 mmol). The mixture was stirred at roomtemperature for 2 hours, and then it was subjected to reversed-phaseHPLC purification to give product L335-M34 as a white solid (0.385 g,99% yield): 1H NMR (500 MHz, DMSO) δ 10.08 (s, 1H), 7.87 (s, 2H), 3.52(s, 2H), 2.42 (s, 3H); ¹³C NMR (DMSO) δ 164.6, 138.9, 130.5, 124.1,121.8, 59.1, 22.6; ESI-HRMS calcd for C₉H₁₀Br₂NO₄S (M+H⁺) 385.8692,found 385.8696.

Kinetic Analysis of mPTPA Inhibition. The phosphatase activity of mPTPAwas assayed using p-nitrophenyl phosphate (pNPP) as a substrate at 25°C. in 50 mM 3,3-dimethylglutarate buffer, pH 7.0, containing 1 mM EDTAwith an ionic strength of 0.15 M adjusted by NaCl. The reaction wasstarted by the addition of 50 L of the enzyme to 150 L of reactionmixture containing pNPP and various concentrations of the inhibitor in a96-well plate. The final concentration for mPTPA was 5 nM. The finalconcentration for pNPP was 1 mM, which was the Km value for mPTPA. Thereaction was quenched after 15 minutes by the addition of 50 L of 5 NNaOH, and then 200 L of reaction mixture was transferred to a 96-wellplate. The non-enzymatic hydrolysis of pNPP was corrected by measuringthe control without the addition of enzyme. The amount of productp-nitrophenol was determined from the absorbance at 405 nm detected by aSpectraMax 384PLUS microplate spectrophotometer (Molecular Devices)using a molar extinction coefficient of 18000 M⁻¹ cm⁻¹. IC₅₀ values werecalculated by fitting the absorbance at 405 nm versus inhibitorconcentration to the following equation:

A ₁ /A ₀ =IC ₅₀/(IC ₅₀ +[I])

where A₁ is the absorbance at 405 nm of the sample in the presence ofinhibitor, A₀ is the absorbance at 405 nm in the absence of inhibitor,and [I] is the concentration of the inhibitor.

For selectivity studies, the PTPs, including mPTPB, PTP1B, TC-PTP, SHP1,SHP2, FAP1, Lyp, PTP-MEG2, HePTP, PTPα, LAR, CD45, PTPg, VHR, Laforin,VHX, Cdc14A, and the low molecular weight PTP, were expressed andpurified from E. coli. The final concentration for all PTPs was 5 nM.The inhibition assay for these PTPs was performed under the sameconditions as mPTPA except using a different pNPP concentrationcorresponding to the Km of the PTP studied. Inhibitor concentrationsused for IC₅₀ measurements cover the range from 0.2 to 5× of the IC₅₀value.

Mycobacterium tuberculosis Strains. The Johns Hopkins Center forTuberculosis Research laboratory reference strain Mtb H37Rv was passagedtwice through mice and frozen in aliquots at −80° C. before use.Aliquots were thawed and grown to logarithmic phase (optical density at600 nm=0.6) in Middlebrook 7H9 broth (Difco Laboratories, Detroit,Mich.) supplemented with 10% OADC (Becton Dickinson), 0.05% Tween, and0.1% glycerol prior to aerosol infection.

Animals. Female guinea pigs (273±20.91 g) with and without jugular veincatheters were purchased from Charles River Laboratories (Wilmington,Mass.). The animals were maintained under specific pathogen-freeconditions and fed water and chow ad libitum. All procedures followedprotocols approved by the Institutional Animal Care and Use Committee atthe Johns Hopkins University School of Medicine.

In Vitro Anti-Mtb Assays. Alamar Blue Assay. A colorimetric,microplate-based Alamar Blue assay (MABA) method was used to determinethe MICs of mPTPA/B against M. tuberculosis isolates. Briefly, cultureswere incubated at 37° C. without shaking in 96-well plates Alamar Bluereagent (Invitrogen) was added at 1:5 v/v prior to readout 24 hourslater using a Fluostar Optima fluorescence plate reader (BMG Labtech),equipped with a 544 nm excitation filter and a 590 nm emission filter.

Macrophage Assays. Inhibition of growth of M. tuberculosis (Erdman andH37Rv) in a macrophage cell culture was assessed as described in Zhou etal., Proc. Natl. Acad. Sci. U.S.A. 107, 4573-4578. Following activationwith 50 U/mL IFN-γ (Sigma, 087k1288), J774 macrophages were infectedwith M. tuberculosis strain at a multiplicity of infection of 1:1 for 1hour, washed, and incubated with 20 g/mL amikacin containing DMEM beforeadding the test compounds. Cells were washed and lysed, and differentdilutions were plated on 7H11 agar plates. Colonies were counted after 3weeks of incubation at 37° C.

Pharmacokinetics and Bioavailability Studies. Separate groups of threecatheterized guinea pigs each were given (i) a single dose of L01-Z08 at20 mg/kg or L335-M34 at 50 mg/kg of body weight (structures shown inTable 11); (ii) L01-Z08 at 20 mg/kg and L335-M34 at 50 mg/kg were giventogether to test for possible drug-drug interactions that might alterthe uptake and/or clearance of one or both of the compounds. All drugswere suspended in 1% DMSO, 0.5% DEA, 48.5% PEG 400, and 50% water. Bloodwas collected for analysis of these drugs in plasma pre-dose and at 1,4, 8, 24, and 48 h post-dose. Plasma was separated and stored at −70° C.until analysis. Plasma drug concentrations were determined by liquidchromatography-mass spectrometry and liquid chromatography-tandem massspectrometry over the concentration range of 0.005-1 mg/L with dilutionto 10 mg/L. Pharmacokinetic variables were calculated from individualdrug concentration-time data using non-compartmental methods asimplemented in WinNonlin version 5.0 (Pharsight, Mountain View, Calif.)as described earlier.

Aerosol Infections. Log-phase cultures of Mtb H37Rv were diluted500-fold (to ˜10⁵ bacilli/mL) in 1× PBS for aerosol infection of guineapigs. A total of 73 guinea pigs were aerosol-infected with a Madisonchamber aerosol generation device (College of Engineering Shops,University of Wisconsin, Madison, Wis.) calibrated to deliverapproximately 10² bacilli into guinea pig lungs, as described in Ahmadet al., J. Antimicrob. Chemother. 65, 729-734.

Antibiotic Treatment. Beginning 28 days after aerosol infection, guineapigs were randomized to different treatment and control groups. Guineapigs were treated 5 days per week for 6 weeks, as indicated in Table 10.Isoniazid (INH, H; Sigma), rifampicin (RIF, R; Sigma), and pyrazinamide(PZA, Z; Sigma) were dissolved in sterile distilled water. A cocktailsolution of HRZ was prepared weekly and kept at 4° C. Aarden compounds(L01-Z08 and L335-M34) were suspended in formulation and stored at roomtemperature for up to 1 week.

TABLE 10 Number of guinea pigs to sacrifice by time-point Regimen Week−4 Day 0 Week 2 Week 4 Week 6 Total Untreated 4 4 4 4 5 21 HRZ 4 4 5 13L01Z08 + 4 4 5 13 HRZ L335M34 + 4 4 5 13 HRZ L01Z08 + 4 4 5 13 L335M34 +HRZ Total 4 4 20 20 25 73 Drugs are to be given at the following dosagesin (mg/kg): Isoniazid (H), 60; Rifampin (R), 1000; Pyrazinamide (Z),300; L01-Z08, 20; L335M34, 50; all drugs given 5 times weekly.

All animals were treated with a formulation consisting of 20% pumpkin(w/v) (Libby's 100% pure pumpkin) mixture supplemented with Vitamin C(50 mg/kg mean body weight) and commercial lactobacillus (BD lactinex)(all purchased from Walmart, Towson, Md.) to improve palatability andhelp stabilize the cecal flora, thereby preventing gastrointestinaldysbacteriosis or antibiotic-associated enteritis. Drug doses wereadministered in a final volume of 0.5 mL and were delivered in theposterior oropharynx by an automatic pipet with a disposable tip.

Study End Points. Guinea pigs were sacrificed on the day after aerosolinfection (day 27), on the day of treatment initiation (day 0), and atthe indicated time points after treatment to determine the numbers ofCFU implanted in the lungs, pretreatment baseline CFU counts, and thepost-treatment CFU counts, respectively.

Animal body weights were recorded on a weekly basis, and lung and spleenweights were recorded at the time of necropsy. The lungs of each animalwere examined at necropsy for grossly visible lesions, and randomsamples from the left caudal lung lobe were dissected, placed into 10%buffered formaldehyde, and paraffin embedded for histopathologicalstaining with hematoxylin and eosin (H&E). At least one entireH&E-stained cross section per animal lung (4 animals/group) was analyzedfor degree of inflammation. The surface area occupied by granulomatousinflammation was determined by ImageJ software-based morphometry ofdigitized images of lung sections, and results are represented aspercentage of lung surface area involved.

The remaining lungs were homogenized in 10 mL of PBS, and homogenateswere plated on 7H11 plates containing cycloheximide (50 mg/L),carbenicillin (100 mg/L), polymyxin B (200000 U/L), and trimethoprim (20mg/L) and incubated for 28 days at 37° C. for CFU enumeration.

Statistical Analysis. CFU data were derived from 4-5 animals per group.Log-transformed CFU were used to calculate means and standarddeviations. Comparisons of data among experimental groups were performedby Student's t test. Group means were compared by one-way analysis ofvariance (ANOVA) with Dunnett's post-test (D0 or untreated controls vstreatment groups) or Bonferroni comparison (all treatment groups), usingGraphPad Prism version 4 (GraphPad, San Diego, Calif.). Values of p<0.05were considered to be statistically significant.

Results and Discussion

Development of Potent and Selective mPTPA and mPTPB Inhibitors.Therapeutic targeting of PTPs has historically been stalled bydifficulties in achieving inhibitor selectivity and bioavailability. Thehighly conserved PTP active site presents considerable challenges inobtaining compounds that can selectively inhibit the target of interestwithout adversely hitting other PTPs. In order to accommodatephospho-substrates, the PTP active site is positively charged, whichfavors negatively charged molecules in high-throughput screeningcampaigns that suffer from poor cell membrane permeability. To addressthe selectivity issue, a novel paradigm was developed for theacquisition of potent and selective PTP inhibitors by targeting both thePTP active site and unique pockets in the vicinity of the active site.To address the bioavailability issue, the existing natural product andFDA-approved drug space was explored for previously unknown PTPinhibitory activities since these molecules already possess acceptablepharmacological properties. Benzofuran salicylic acid was previouslyidentified as a privileged pharmacophore for mPTPB. Using afragment-based medicinal chemistry approach, the benzofuran salicylicacid core was transformed herein into a highly potent and selectivemPTPB inhibitor (L01-Z08, Table 11) with excellent in vivo efficacy.

TABLE 11 Molecular and Cellular Properties of Lead mPTPA and mPTPBInhibitors In vitro anti-Mtb activity (uM) Biochemical Fold selectivityMABA- potency against vs. MIC^(b) target (IC₅₀, nM) vs. PTP H37RvMtb-infected Structure Name Target mPTPA mPTPB mPTPA/B panel^(a) Erdmanmacrophages^(c)

L335M34 mPTPA  160 >3200 >20 >20 >10 >10 1.38

L01Z08 mPTPB 2500 38 66 >37 >10 >10 <5 ^(a)Human PTP panel: PTP1B,TC-PTP, SHP1, SHP2, FAp1, Lyp, Meg2, HePTP, laforin, VHX, VHR, LMWPTP,Cdc14A, PTPα, LAR, CD45, PTPRG. ^(b)MABA-MIC = microplate Alamar Blueassay for minimum inhibitory concentration. ^(c)IC₉₀ in macrophagesactivated with interferon-γ.

More recently, it was discovered that cefsulodin, a third generationcephalosporin β-lactam antibiotic, exhibits inhibitory activity againsta number of PTPs. Fragmentation analysis of cefsulodin identifiedα-sulfophenylacetic amide (SPAA) as an mPTP-inhibiting pharmacophore anda novel pTyr mimetic. Structure-guided and fragment-based optimizationof SPAA led to compound L335-M34, which displayed an IC₅₀ value of 160nM for mPTPA (Table 11). Kinetic analysis revealed that L335-M34 was areversible and competitive inhibitor of mPTPA with a Ki of 56±2.0 nM(FIG. 7). To determine the specificity of L335-M34, its inhibitoryactivity toward mPTPB and a panel of mammalian PTPs was measured,including cytosolic PTPs, PTP1B, TC-PTP, SHP1, SHP2, FAP1, Lyp,PTP-Meg2, and HePTP, the receptor-like PTPs, PTPα, LAR, CD45, and PTPRG,the dual specificity phosphatases VHR, Laforin, VHX, and Cdc14A, and thelow molecular weight PTP. As shown in Table 11, L335-M34 was highlyselective for mPTPA, exhibiting greater than 20-fold selectivity overall PTPs examined. L335-M34 appears to represent the most potent andspecific mPTPA inhibitor reported to date.

Cellular Activity of mPTPA and mPTPB Inhibitors L335-M34 and L01-Z08.The mPTPA inhibitor L335-M34 was highly selective for its target, withan IC₅₀ of 160 nM against mPTPA, but no significant activity againstmPTPB or a panel of human PTPs at concentrations below 3 μM. BecausemPTPA is a secreted virulence factor that regulates host antibacterialresponses rather than Mtb physiology, it was unsurprising that L335-M34was devoid of activity in standard MIC assays; however, the compound wasable to markedly decrease bacterial load in Mtb-infected macrophages atlow micromolar concentrations (Table 11).

The L01 family comprises three highly active and selective mPTPBinhibitors. The selected lead compound from this series, L01-Z08,displayed a potency of 38 nM against mPTPB and was 66-fold less potentagainst mPTPA and at least 37-fold selective when screened against apanel of 17 human PTPs. Like the mPTPA inhibitor L335-M34, L01-Z08 wasinactive in the MIC assay but displayed potent anti-Mtb activity inJ774A.1 macrophages (Table 11).

mPTPA and mPTPB Inhibitors Are Bioavailable and Well-Tolerated in GuineaPigs Following Oral Dosing. As shown in FIG. 8, L01-Z08 and L335-M34showed good oral bioavailability and half-life in guinea pigs (Table12). Both drugs were rapidly absorbed, reaching peak concentrationswithin a few hours with a typical biphasic plasma clearance curve.Because the drugs were to be co-formulated for oral delivery in thetherapy study, a combination PK study was performed to confirm that theywere amenable to co-administration. Co-formulating L01-Z08 with L335-M34in a single dosing solution did not negatively affect uptake orclearance of either drug. In fact, the bioavailability of L335-M34 wasaffected only moderately; a slight increase in uptake rate led to agreater peak concentration, which was offset by somewhat more rapidclearance, so that the overall exposure (AUC_(ALL)) was essentiallyunchanged. By contrast, L01-Z08 exposure was enhanced byco-administration (both C_(max) and beta-phase T_(1/2) were elevated),but a reduction in the volume of distribution suggested that drugdelivery to the tissues was probably not improved (Table 12). The PKstudy strongly indicated that the two compounds could be delivered withadequate efficiency in the guinea pig by the oral route. As shown in thebottom panel of FIG. 8, at the doses selected for use in the efficacystudy, L335-M34 and L01-Z08 were detected at concentrations 10-fold inexcess of their biochemical IC₅₀ values for 12-14 and 20-24 hours,respectively, suggesting that once daily oral dosing was an appropriateschedule for each drug. However, it should be noted that a higher degreeof selectivity for these compounds was observed in the biochemicalassays (IC₅₀) than in the whole-cell assays in macrophages (growthinhibition/IC₉₀), perhaps due to cell permeability issues.

TABLE 12 Pharmacokinetics of mPTPA and mPTPB Inhibitors in GuineaPigs^(a) Cmax AUC_(ALL) Clearance (ng/mL) Compound Dose type (h · ng/mL)(mL/h/kg) T_(max) ^(b) (h) Tmax^(b) (h) Half-life (h) V_(D) ^(c) L335M34Single 54406.61 917.462 5142.47 2.5 5.197 6878.467 L335M34 Co-admin^(d)52752.61 950.034 7064.956 2.5 4.16 5702.19 L01Z08 Single 13166.891518.474 1870.43 1.587 5.512 12074.939 L01Z08 Co-admin^(d) 28161.76701.564 3059.668 1.587 6.141 6215.132 ^(a)Data represent mean values for2-3 animals. ^(b)T_(max) is the time required to achieve the maximalconcentration (C_(max)). ^(c)Volume of distribution. ^(d)Both compoundsL335M34 (50 mg/kg) and L01Z08 (20 mg/kg) were co-administered orally.

Guinea pigs receiving L01-Z08 20 mg/kg and L335-M34 50 mg/kg once dailyalone or in combination for 6 weeks showed no overt signs of toxicityand displayed similar mean weight gain to those receiving HRZ (FIG. 9).All guinea pigs receiving L01-Z08 and L335-M34 survived and gainedweight throughout the course of the efficacy study.

Dual Inhibition of mPTPA and mPTPB Significantly Reduces Guinea Pig LungBacillary Burden Relative to HRZ Alone. Since each mPTP modulatesdistinct Mtb clearance pathways in macrophages, it was hypothesized thatdual inhibition of mPTPA and mPTPB would enhance the bactericidalactivity of the standard anti-tubercular regimen in guinea pigs morethan adjunctive therapy with either agent alone. Following aerosolinfection of guinea pigs with Mtb H37Rv, 2.06±0.15 log₁₀ bacilli weredeposited in the lungs on day −27, and the organisms multiplied to apeak burden of 6.11±0.15 log₁₀ CFU (colony-forming units) on day 0 (timeof treatment initiation). Thereafter, bacillary growth was controlled inthe lungs of untreated guinea pigs, which had 5.82±0.17 log₁₀ CFU in thelungs at the conclusion of this Example.

Following 2 weeks of treatment, all guinea pigs in the HRZ, HRZ+L335-M34(A), HRZ+L01-Z08 (B), and HRZ+AB groups were able to contain Mtbmultiplication in the lungs, resulting in mean bacillary burdens of4.44±0.31, 4.07±0.15, 4.15±0.17, and 3.77±0.21 log₁₀, respectively.After 2 weeks of treatment, lung CFU counts in animals treated withHRZ+AB were significantly (p<0.01) lower than those treated with HRZ(FIG. 10A). However, no significant differences were observed in lungCFU between HRZ+A, HRZ+B, and HRZ +AB.

At 4 weeks post-treatment, a similar trend was seen and the hierarchy ofbactericidal activities of the various regimens was HRZ+AB (1.15±0.17log₁₀ CFU)>HRZ+B (1.64±0.19 log₁₀ CFU)>HRZ+A (1.65±0.2 log₁₀ CFU)>HRZ(1.70±0.12 log₁₀ CFU) (FIG. 10B). HRZ+AB was significantly more activethan HRZ+B, HRZ+A, and HRZ alone (p<0.001).

After 6 weeks of treatment, HRZ reduced mean lung CFU by 4.27 log₁₀compared to untreated controls (FIG. 8). The addition of L335-M34 (A) orL01-Z08 (B) to the standard regimen further reduced mean lung CFU by0.14 log₁₀ and 0.17 log₁₀, respectively, and the combination (AB)lowered mean lung CFU by 0.45 log 10 (p<0.0001) relative to HRZ (FIG.10C).

The gross pathology of guinea pig lungs (data not shown), as well asmean guinea pig lung and spleen weights (FIGS. 11A-11C), correlated withthe efficacy of the various chemotherapy regimens. Interestingly, themean lung surface area involved by inflammation after 6 weeks oftreatment was significantly lower in the HRZ+AB (9.23%) group relativeto the HRZ group (36.28%) (p=0.028). These results suggest that thiseffect on improved histopathology is primarily conferred by inhibitionof mPTPA (HRZ+A vs HRZ+AB, p=0.68) (FIGS. 12 & 13).

The fragment-based lead optimization strategies have yielded twocompounds, L01-Z08 and L335-M34, with potent activity againstintracellular Mtb, as well as favorable PK and toxicity profiles.L01-Z08 and L335-M34 are inhibitors of the key secreted Mtb enzymes,mPTPB and mPTPA, respectively, and thus provide a novel mechanism ofaction for the treatment of TB. Both phosphatases are secreted by Mtbinto the cytoplasm of the macrophage and are important for persistenceof mycobacterial infection. In order to determine the potential fortranslation of these findings to the clinical arena, whether mPTPinhibitors could be beneficial as an adjunctive treatment when combinedwith the standard first-line regimen against drug-susceptible TB wasevaluated in guinea pigs. The two mPTPA and mPTPB inhibitor leadcompounds showed promising oral bioavailability and tolerability in thismodel. Although each inhibitor alone added little bactericidal activityto the standard regimen, dual inhibition of mPTPA and mPTPBsignificantly reduced the lung bacillary burden relative to HRZ at eachtime point studied.

PTKs are the molecular targets for a growing number of anticanceragents; however, there is a notable absence of drugs targeting the PTPs.Although many disease-relevant pathways are also controlled by PTPs, thelatter have proven to be exceptionally challenging targets for thedevelopment of new therapeutic agents, due primarily to the poorbioavailability of existing PTP inhibitory compounds. The observed oralbioavailability and in vivo efficacy of L01-Z08 and L335-M34 arepromising and further demonstrate that it is feasible to obtain PTPinhibitors that are sufficiently polar to bind the active site and yetstill possess favorable pharmacological properties for therapeuticdevelopment.

Given the unique mechanisms of action of the mPTPA and mPTPB inhibitors,these compounds are expected to provide additive bactericidal activityto the standard regimen for drug-susceptible TB as well as to novelregimens for drug-resistant TB. Moreover, concomitant treatment withsuch inhibitors may reduce the risk for selection of strains resistantto currently available anti-TB drugs during treatment. Previous work hasshown that small-molecule inhibitors of both mPTPA and mPTPB are capableof reducing intracellular mycobacteria in infected macrophages. It isinteresting to note that adjunctive inhibition of mPTPA led to improvedlung histopathology relative to standard treatment alone. A recent studyshowed that mPTPA dephosphorylates a second substrate, glycogen synthasekinase-α (GSK-α), causing its activation and the subsequent inhibitionof the cell death program in infected macrophages. Based on availabledata, dual inhibition of mPTPA and mPTPB appears to undermine Mtbinfection by (i) increasing intracellular destruction of bacteria, (ii)promoting host-beneficial apoptosis of infected macrophages, and (iii)increasing host immunologic awareness of, and responsiveness to, Mtbinfection.

Previous studies have indicated that mPTPA is not essential for Mtbsurvival in mice, implying that the murine model fails to recapitulatethe phenotypes reported in human macrophages. Although the mouse modelhas long been used to evaluate TB drugs, it has been increasinglyrecognized in the TB field that observations made in mice are notpredictive of treatment outcomes in human clinical trials, nor is early“sterilization” a predictor of cure. In the current Example, thewell-characterized guinea pig model of TB chemo-therapy was used.Compared to mice, guinea pig TB granulomas more closely approximatetheir human counterparts with respect to cellular composition, granulomaarchitecture, and the presence of caseation necrosis. In addition,tissue hypoxia is present in guinea pig TB granulomas, but absent inmouse TB lung lesions. These histological and micro-environmentalfactors, which may be biological determinants of Mtb persistence, aswell as concordance of treatment outcomes with those of recent humanstudies, make the guinea pig model an attractive one for testing theactivity of novel anti-tubercular agents. However, the anti-tubercularactivity of these agents could be further characterized in otherclinically relevant models, such as the rabbit and non-human primate.

The data support the further development of the Mtb PTP inhibitor classof drugs. PTP inhibitors lack direct antimicrobial activity, but promoteintracellular Mtb killing in vitro. These findings suggest a modestincrease in killing by the standard regimen with dual mPTPA/Binhibition, as well as a favorable PK interaction between the agents.Significantly, the data suggest that PTP inhibitors may improve clinicaloutcomes by ameliorating lung pathology.

Example 4

Selective inhibition of LMWPTP. SPAA based compounds from the librarieswere studied as inhibitors against a panel of PTPs, including LMWPTP andLaforin. In the library, 8 compounds exhibited a similar level ofactivity against human LMPTP and Laforin (IC₅₀ about 5-10 μM), generallywith at least about 10-fold selectivity for LMPTP and Laforin over otherPTPs (Tables 5 and 13).

TABLE 13 IC₅₀ values of LMWPTP inhibitors against a panel of PTPs IC₅₀(μM) LMWPTP SHP2 PTP1B SHP1 TcPTP VHX STEP HePTP TbPTP FAP-1 VHR LYPSsu72 PEST Laforin L319M34 5.3 >100 >200 55.5 >10092.0 >200 >100 >100 >100 >200 >200 >100 72.3 4.05 L319M50 12.1 >200 >20053.2 >100 >100 >200 >100 >100 73.0 >200 >200 66.6 69.9 2.06 L319M545.6 >200 >200 >200 >200 >200 >200 >200 >200 >100 >200 >200 >200 >2004.25 L319M637.7 >100 >100 >200 >100 >200 >100 >100 >100 >100 >100 >100 >100 >1002.65 L319M688.1 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >2007.47 L319M887.3 >100 >100 >200 >100 >200 >100 >100 >100 >100 >100 >100 >100 >1008.51 L319N15 7.1 >100 >200 59.6 >200 >100 >100 50.0 >200 34.0 >200 >20069.0 43.5 2.1 L319N22 6.4 >200 >200 >200 >200 >200 >100 94.0 >20076.0 >200 >200 83.0 66.9 27.0

To develop specific inhibitors of either LMPTP or Laforin, L319N15 (witha benzo[d]thiazol-2-yl moiety attached to the amide group, Table 5) wasselected as a lead compound in a medicinal chemistry effort. As shown inTable 6, 22 analogues of L319N15 were synthesized, including L319N13(with a thiazole-2-yl moiety), L319N14 (with a 4-phenylthiazole-2-ylmoiety), L319N15-01 to L319N15-13 (with various substituents on thebenzo[d]thiazol-2-yl moiety of L319N15), L319N15-14 to L319N15-18 (withvarious substituents on the α benzene ring), L335N15, L335N15-07, andL338N15 (with the deletion of α benzene ring). The IC₅₀ of thesecompounds were measured against LMPTP and Laforin (Table 14).

TABLE 14 IC₅₀ values of L319N15 analogues against LMWPTP andrepresentative PTPs IC₅₀ (μM) LMWPTP SHP2 PTP1B LYP CD45 Laforin L319N13100 >100 >100 >100 >100 8 L319N14 20 31 160 15 70 6 L319N15 8.6 160 >200190 180 2.1 L319N15-01 4.6 41 70 22 100 2.2 L319N15-02 9.6 129 >200110 >200 4 L319N15-03 7.6 >200 >200 >200 >200 1.2 L319N15-04 4.8112 >200 90 >200 1.1 L319N15-05 4.2 173 >200 150 >200 1.7 L319N15-06 5.630 80 23 130 2.3 L319N15-07 2.1 >200 >200 >100 >200 >200 L319N15-088.2 >200 >200 >200 >200 2.0 L319N15-09 5.3 47 160 20 80 3.2 L319N15-105.8 >200 >200 >200 >200 2.1 L319N15-11 3.03 55 200 28 70 1.7 L319N15-1214 190 >200 166 160 3.3 L319N15-13 70 83 >200 31 90 >200L319N15-14 >200 >200 >200 >200 >200 19 L319N15-15 21 2.6 31 5 30 4.3L319N15-16 >200 135 >200 180 >200 66 L319N15-17 >100 >100 >100 >100 >10014 L319N15-18 >100 >100 >100 >100 >100 32 L335N152.1 >100 >100 >100 >100 >100 L335N15-07 0.3 >100 >100 >100 >100 >100L338N15 30 NA NA NA NA NA

As shown in Table 14, most of the L319N15 analogues preferably inhibitLaforin (IC₅₀ about 1 to 10 μM). Remarkably, L319N15-07, L319N15-13,L335N15, and L335N15-07 showed specificity towards LMPTP, but notLaforin. For example, under the same assay conditions, L319N15-07 andL335N15-07 inhibited LMPTP at IC₅₀ values of 2.1 μM and 0.3 μM,respectively, while they showed no detectable inhibition against Laforinat 100 μM. Similarly, L319N15-13 showed an IC₅₀ value of 70 μM againstLMPTP, did not inhibit Laforin at 200 μM.

Remarkably, these three compounds all have a polar substituent atposition 3 on the benzene ring of the benzo[d]thiazol-2-yl moiety: anitro group for L319N15-07 and L335N15-07, and a carboxy group forL319N15-13 (Table 6). Without being bound by any theory, it ishypothesized that the negative or partially negative charge (such asthat in the nitro and the carboxy groups) in the amide substituent (suchas the benzo[d]thiazole moiety of L319N15) causes these compounds topreferably inhibit LMWPTP over Laforin.

Surprisingly, L319N15-07 and L335N15-07 exhibited no detectableinhibition against any PTP in Table 8 (other than mPTPA and mPTPB) at upto 100 μM (see Table 10 for results of SHP2, PTP1B, LYP, and CD45).These data indicate that L319N15 analogs inhibit LMPTP more specificallyover the other 30 PTPs tested. In particular, for L319N15-07 andL335N15-07, the inhibition is more than 100-fold selective for LMWPTPover other PTPs (Table 14). Thus, the SPAA based L319N15 analogs can beused to develop anti-cancer and anti-diabetes drugs targeting at LMWPTP.

Example 5

Potent and specific inhibition of mPTPA. Several potent inhibitorsagainst mPTPA have been identified in the libraries described herein(Table 4). Results of IC₅₀ measurements against mPTPA and other PTPs areshown in Table 15.

TABLE 15 IC₅₀ values of mPTPA inhibitors against a panel of PTPs IC₅₀(μM) L319M34 L319M54 L319M68 L319M88 L319N15 L319N22 L335N15 mPTPA 0.20.27 0.25 0.143 0.194 0.972 0.263 mPTPB 0.055 0.078 5.0 0.282 0.1160.203 9.5 LMWPTP 4.5 5.0 20 6.1 7.1 6.39 2.1 Laforin 1.05 4.25 7.5 8.52.1 50 >100 YopH >200 >200 >200 >200 >200 >200 >100TbPTP1 >200 >200 >200 >200 >200 >200 >100PTP1B >200 >200 >200 >200 >200 >200 >100TcPTP >200 >200 >200 >200 >200 >200 >100LYP >200 >200 >200 >200 >200 >200 >100 PEST 42.3 >200 >200 >200 43.567 >100 SHP1 >200 >200 >200 >200 >200 >200 >100SHP2 >200 >200 >200 >200 >200 >200 >100 HePTP >200 >200 >200 >200 5094 >100 Meg2 >200 >200 >200 >200 >200 >200 >100PTPH1 >200 >200 >200 >200 >200 >200 >100 FAP1 >200 180 >200 >200 3476 >100 CD45 >200 >200 >200 >200 >200 >200 >100LAR >200 >200 >200 >200 >200 >200 >100PTPα >200 >200 >200 >200 >200 >200 >100PTPβ >200 >200 >200 >200 >200 >200 >100PTPγ >200 >200 >200 >200 >200 >200 >100PTPσ >200 >200 >200 >200 >200 >200 >100PTPε >200 >200 >200 >200 >200 >200 >100PTPμ >200 >200 >200 >200 >200 >200 >100 STEP >200 >200 >200 >200 >200140 >100 MKP3 >200 >200 >200 >200 >200 >200 >100VHR >200 >200 >200 >200 >200 >200 >100 VHX92 >200 >200 >200 >200 >200 >100 VHX >200 >200 >200 >200 >200 >200 >100CDC14A >200 >200 >200 >200 >200 >200 >100PP5 >200 >200 >200 >200 >200 >200 >100 Ssu72 >200 >200 >200 >200 6983 >100 IC₅₀ (μM) L335N15-07 L335M34 L335M54 L335M68 L335M88 mPTPA NA0.16 0.65 2.2 3.0 mPTPB NA 3.1 NA NA 55 LMWPTP 0.3 4.2 1.86 63   12Laforin >100 >100 >100 NA >100 YopH >100 >100 >100 NA >100TbPTP1 >100 >100 >100 NA >100 PTP1B >100 >100 >100 NA >100TcPTP >100 >100 >100 NA >100 LYP >100 >100 >100 NA >100PEST >100 >100 >100 NA >100 SHP1 >100 >100 >100 NA >100SHP2 >100 >100 >100 NA >100 HePTP >100 >100 >100 NA >100Meg2 >100 >100 >100 NA >100 PTPH1 >100 >100 >100 NA >100FAP1 >100 >100 >100 NA >100 CD45 >100 >100 >100 NA >100LAR >100 >100 >100 NA >100 PTPα >100 >100 >100 NA >100PTPβ >100 >100 >100 NA >100 PTPγ >100 >100 >100 NA >100PTPσ >100 >100 >100 NA >100 PTPε >100 >100 >100 NA >100PTPμ >100 >100 >100 NA >100 STEP >100 >100 >100 NA >100MKP3 >100 >100 >100 NA >100 VHR >100 >100 >100 NA >100VHX >100 >100 >100 NA >100 VHX >100 >100 >100 NA >100CDC14A >100 >100 >100 NA >100 PP5 >100 >100 >100 NA >100Ssu72 >100 >100 >100 NA >100

Particularly, L319M68 was shown to inhibit mPTPA with IC₅₀ at 0.25 μM, a20-fold selectivity over mPTPB, a 80-fold selectivity over human LMWPTP,a 30-fold selectivity over Laforin, and a >800-fold selectivity over allother human PTP tested. Kinetic studies show that L319M68 is acompetitive inhibitor against mPTPA with Ki at 0.13 μM. In addition,L335M34 showed an IC50 at 0.16 μM, a 20-fold selectivity over mPTPB, a30-fold selectivity over human LMWPTP, and a >1000-fold selectivity overall other human PTP including laforin. Hence, these compounds are potentand selective inhibitors of mPTPA, allowing for development of anti-TBagents targeting at mPTPA.

Example 6

SPAA based mPTPA and mPTPB dual inhibitors. Compounds inhibiting mPTPAand mPTPB are also desirable in developing anti-TB agents. The approachis particularly attractive, given that mPTPA and mPTPB are produced andsecreted by the same Mtb strain.

Compounds L319M34, L319M54, and L319N15 described herein (Table 1) havebeen identified to inhibit both mPTPA and mPTPB (IC₅₀ approximately0.05-0.2 μM), but not other PTPs (see Table 15). For example, L319M34demonstrated an IC₅₀ of 0.2 and 0.055 μM to mPTPA and mPTPB,respectively, with a selectivity of at least 20-fold over any othermammalian PTPs, including its close analogue, human LMWPTP. Theseresults indicate that SPAA based compounds can be used as dualinhibitors for both mPTPA and mPTPB, allowing for the development ofanti-TB agents targeting at both PTPs.

Example 7

Potent and specific inhibition of Laforin. From the SPAA based libraryof compounds described herein, several potent and specific Laforininhibitors have been identified (Table 2). The IC₅₀ concentrations ofthese compounds range from low μM to nM scale (Table 16).

TABLE 16 IC₅₀ values of Laforin inhibitors against a panel of PTPs IC₅₀(μM) Laforin LMWPTP SHP2 PTPIB SHP1 TcPTP VHX STEP HePTP TbPTP-1 FAP-1VHR LYP Ssu72 PEST L319M50 2.06 12.1 >200 >20053.2 >100 >100 >200 >100 >100 73.0 >200 >200 66.6 69.9 L319M52 5.0872.3 >200 >200 62.3 >100 >100 >200 >100 >100 88.0 >200 >200 97.6 77.4L319M63 2.657.7 >100 >100 >200 >100 >200 >100 >100 >100 >100 >100 >100 >100 >100L319M373- 1.3 10.2 NA NA NA NA NA NA NA NA NA NA NA NA >200 2 L319M792.2 23.6 NA NA NA NA NA NA NA NA NA NA NA NA 26.5 L319N08 4.7228.9 >100 >200 64.6 >200 >200 >100 78.0 >100 71.0 >200 >200 72.9 59.7L319N72 4.8 98.1 NA NA NA NA NA NA NA NA NA NA NA NA >200 L319-2-9 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200 >200A60 L319-05- 0.8 29 69 183 200 96 100 >100 150 93 70 50 62 170 160 A64L319-21- 0.14 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50M06 L319-21- 1.11 >50 51 >50 >50 >50 >50 >50 >50 >50 39 >50 >50 >50 >50M50 L319-21- >0.1 NA NA NA NA NA NA NA NA NA NA NA NA NA NA M51 L319-21-0.15 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 M52

As shown in Table 16, L319M73-2 inhibited Laforin at an IC₅₀ value of1.3 μM, with an 8-fold specificity for Laforin over LMPTP and a 100-foldspecificity over 30 other human PTPs. In addition, L319-02-A60,L319-05-A64 (IC₅₀=9, 0.8 μM) are highly specific with >20-foldselectivity against any other PTPs, including LMWPTP.

Remarkably, L319-21 series compounds (L319-21-M06, -M50, -M51, and -M52)inhibited Laforin at IC₅₀ around 0.1 μM, with an over 500-foldselectivity over other PTPs. Thus, SPAA based compounds described hereinare potent and specific PTP inhibitors, which can be used to study themolecular mechanisms of Lafora disease.

Example 8

Inhibition of SHP2. Several compounds in the SPAA based librarydescribed herein (for example, L319-20-M24, L319-20-M74, L319-20-M78,L319-20-N08, L319-20-N25, L319-22-M34, L319-22-M50, L319-22-M51,L319-23-M74, L319-24-M74, L319-24-M77, and L319-04-B34) have beenidentified as effective inhibitors of SHP2 (Tables 3 and 17). The IC₅₀of these compounds for SHP2 inhibition was shown to be approximately inthe 1-10 μM range.

TABLE 17 IC₅₀ values of SHP2 inhibitors. SHP2 Compound ID IC₅₀, μML319-20-M24 1.5 L319-20-M74 0.9 L319-20-M78 0.7 L319-20-N08 1.4L319-20-N25 2.3 L319-22-M34 5.7 L319-22-M50 7.5 L319-22-M51 10L319-23-M74 3.5 L319-24-M74 1.34 L319-24-M77 1.1 L319-04-B34 7.9

Example 9 Experimental Section

Protein expression and purification. The SHP2 catalytic domain (residues224-528) used for kinetic studies was cloned into the pET-21a+ vectorusing NdeI and XhoI (NEB), and mutants E508A and R362A were generatedusing QuikChange mutagenesis Kit (Stratagene). These proteins wereexpressed in E. coli BL21(DE3) and purified with Ni-NTA resin (Qiagen).The SHP2 catalytic domain (residues 262-528) used for crystallizationwas expressed in E. coli BL21(DE3) and purified by Ni-NTA agarose(Qiagen) followed by sequential chromatography of HiPrep 26 desaltingcolumn (GE Healthcare), and cation exchange column packed with SPsepharose (GE Healthcare). Protein purity was determined to be greaterthan 95% by SDS-PAGE and Coomassie blue staining.

Enzyme kinetics and inhibition studies. The SHP2 phosphatase activitywas assayed in 96-well plates using pNPP as a substrate at 25° C. in 50mM 3,3-dimethylglutarate (DMG) buffer (pH=7.0), containing 1 mM EDTAwith an ionic strength of 0.15 M adjusted by NaCl. The FDA-approved drugcollection or the SPAA based libraries were screened in a 96-well formatat two different compound concentrations (10 and 20 μM for FDA drugs and10 and 1 μM for the SPAA libraries). The reaction was initiated by theaddition of 50 μl of SHP2 to 150 μl of reaction mixture containing pNPPand the compound, the resulting 200 μl mixture has SHP2 at a finalconcentration of 20 nM, and pNPP at a final concentration of 3 mM (theK_(m) for pNPP). The reaction was allowed to proceed for 10 minutes, andthen quenched by addition of 50 μl of 5N NaOH. The amount of product,p-nitrophenol, was determined from the absorbance at 405 nm detected bya Spectra MAX340 microplate spectrophotometer (Molecular Devices) usinga molar extinction coefficient of 18,000 M⁻¹ cm⁻¹. The nonenzymatichydrolysis of pNPP was corrected by measuring the control withoutenzyme. The Michaelis-Menten kinetic parameters k_(cat) and K_(m) weredetermined from a direct fit of the velocity versus substrateconcentration data to Michaelis-Menten equation using SigmaPlot program.Inhibitor concentrations used for IC₅₀ measurements cover the range from0.2 to 5× of the IC₅₀ value. IC₅₀ values for cefsulodin and compounds2-7 were calculated by fitting the absorbance at 405 nm versus inhibitorconcentration to the following equation:

A ₁ /A ₀ =IC ₅₀/(IC ₅₀ +[I])

where A₁ is the absorbance at 405 nm of the sample in the presence ofinhibitor; A₀ is the absorbance at 405 nm in the absence of inhibitor;and [I] is the concentration of the inhibitor.

For inhibitor selectivity profiling studies, the PTPs, includingcytosolic PTPs, SHP1, PTP1B, LYP, HePTP, and PTP-Meg2, the receptor-likePTPs, CD45, PTPβ, PTPε, PTPγ, PTPμ and LAR, the dual specificityphosphatases VHR and CDC14A, the low molecular weight (LMW) PTP, and theSer/Thr protein phosphatase PP5, were expressed and purified from E.coli. The inhibition assays for these PTPs were performed under the sameconditions as SHP2 except using a different pNPP concentrationcorresponding to the K_(m) of the PTP studied.

Crystallography studies. The SHP2●cefsulodin co-crystals were grown at20° C. in the hanging drops containing 1.5 □L protein solution (8 mg/mlSHP2 with 1 mM cefsulodin in 20 mM MES, pH 5.8, 300 mM NaCl, 2 mM DTTand 1 mM EDTA) and 1.5 □L reservoir solution (20% PEG3350, 33 mM citricacid, 67 mM BIS-TRIS propane, pH=7.4). The crystals were transferredinto 2 μL of cryo-protectant buffer (150 mM NaCl, 1 mM cefsulodin, 30%PEG3350, 33 mM citric acid, 67 mM BIS-TRIS propane, pH=7.4), allowed tosoak for 5 minutes and then flash-frozen by liquid nitrogen. Data werecollected at the 19-BM beam line at the Advanced Photon Source (APS) andwere processed with HKL3000. The data were collected to 1.6 A resolutionin the P₂₁ space group. The phase was determined by molecularreplacement with Molrep (Vagin, A.; Teplyakov, A. J. Appl. Crystallogr.1997, 30, 1022) using the coordinates of reported SHP2 structure (PDBID:3B7O) as the search model. The SHP2●cefsulodin complex structure wasrefined using phenix.refine in the PHENIX software suite. The electrondensity maps were inspected and the model was tuned in Coot. The datacollection and structure refinements statistics are summarized in Table19.

Molecular modeling of the interaction of SHP2 with cefsulodin.AutoDock_Vinal.1.2 program (Trott, O.; Olson, A. J. J. Comput. Chem.2010, 31, 455) was used to build the most likely binding modes forcefsulodin and SHP2. The 3D-structure of cefsulodin was modeled andenergy-minimized in Chem3D program, and the coordinates of SHP2catalytic domain (PDBID: 3B7O) were retrieved from the Protein DataBank. Both ligand and the receptor structures were pre-processed inAutoDockTools1.5.4, such as merging non-polar hydrogens, addingGasteiger charges, setting rotatable bonds for the ligand, addingsolvation parameter for the receptor, and so on. A docking space of21.0×21.0×22.5 Å was visually set around the catalytic active site, theparameter of exhaustiveness, num_modes and energy_range was set to 20,200 and 4 respectively, and the default values were used for the otherparameters.

Characterization of cefsulodin-mediated SHP2 inhibition by Q-TOF ESI-MS,LC-MS, and HPLC. Agilent 6520 Accurate-Mass was used for the Q-TOFESI-MS studies. Water and acetonitrile (both containing 0.1% formicacid) were used as eluent (80% water, flow 50 μL/min, run time 5minutes). ESI positive mode was used with mass range from 300 to 1700,the gas temperature was 325° C., and vaporizer temperature was: 819° C.The sample was adjusted to have a concentration at 0.1 mg/mL, theinjection volume of each sample was 1 μL. Mass hunter software was usedfor protein deconvolution data analysis to calculate the molecularweight of the protein.

Agilent Technologies 6130 quadrupole LC-MS instrument was used for theLC-MS studies. A C18 reserved phase column (phenomenex, 50×4.6 mm) wasused as stationary phase, water and methanol (both containing 0.1%formic acid) were used as mobile phase (gradient: 0-100% methanol, flow0 8 ml/min, run time 15 minutes), and UV absorbance at the fixedwavelength of 254 nm and positive and negative ESI-MS data wererecorded. The sample was adjusted to have a concentration at 0.1 mg/mL,and the injection volume of each sample was 0.2 μL. The retention timeand corresponding ESI-MS data were used as identity of molecules.

Waters 2545 preparative HPLC purification system was used for compoundpurification. A C18 reserved phase column (Sunfire, 50×150 mm) was usedas stationary phase, water and methanol (both containing 0.1%trifluoroacetic acid) were used as mobile phase (gradient: 0-100%methanol, flow 50 ml/min, run time 60 minutes), and UV absorbance at thefixed wavelength of 254 nm was used for fraction collection. Thecompound identity was validated by LC-MS studies following HPLCpurification.

Cell proliferation and immunoblot analysis. Human non-small cell lungcarcinoma cell line H1975 was cultured at 37° C. and 5% CO₂ in RPMI-1640(Corning) supplemented with 10% fetal bovine serum (HyClone). Humanbreast cancer cell line MDA-MB-231 was cultured in DMEM supplementedwith 10% fetal bovine serum. For cell proliferation assay, 2-3×10³ cellswere seeded in each well of 96-well plates. After treating cells withcompounds for 2 days, cells were incubated with 50 □g/ml MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) for ˜3-4hours. Then, the culture medium was removed, DMSO was added to dissolvethe formazan crystals. Wells containing only media were used forbackground correction. The optical density was measuredspectrophotometrically at 540 nm. For signaling analysis, cells wereserum-starved overnight followed by treatment with vehicle or compoundsfor 3 hours, and then either left un-stimulated or stimulated with 5μg/ml EGF or 10 nM PMA (Sigma) for indicated time. Cells were lysed andthe lysates were electrophoresed on a 10% polyacrylamide gel, and thentransferred to a nitrocellulose membrane, and probed withanti-phospho-ERK1/2 and anti-ERK1/2 (Cell Signaling) antibodies followedby incubation with horseradish peroxidase-conjugated secondaryantibodies. The blots were developed by the enhanced chemiluminescencetechnique using the SuperSignal West Pico Chemiluminescent substrate(Pierce).

SHP2 inhibitor 2 effects on cell growth in Matrigel. Approximately300,000 SKBR3 cells were seeded into 150 □L of growth factor reducedMatrigel (BD) that was then covered with 2 mLs of media containingeither 20 □L vehicle (DMSO) or the indicated concentrations of compound2. Cells were then imaged at 24-hour intervals using a NIKON SMZ1500stereomicroscope. After 5 days the cells were recovered from theMatrigel by the following method. Media was aspirated and cells werewashed with 500 □L of cold PBS. 150 □L of RIPA lysis buffer supplementedwith ProteCEASE-50. EDTA Free protease inhibitors (GBiosciences) wasadded to the Matrigel. Cells were scraped into a slurry and instantlyfrozen using dry ice for 5 minutes. After thawing tubes were spun downat 4° C. for 10 minutes at 14K rpm. Proteins were then resolved bySDS-PAGE and the relative levels of total and phospho-ERK1/2 weredetected by immunoblot analysis.

Chemical synthesis: materials and general procedures. Cefsulodin andother antibiotics were used as purchased from Sigma-Aldrich, and allother reagents were from Fisher Scientific. ¹H and ¹³C NMR spectra wereobtained on a Bruker Avance II 500 MHz or a Bruker Fourier 300 MHz NMRspectrometer with TMS or residual solvent as standard. Accurate massdata was obtained using an Agilent 6520 Accurate-Mass instrument.

Synthesis of Lib-1, Lib-2, Lib-3, and Lib-4. To α-sulfophenyl acetylchloride (0.234 g, 1 mmol) and DIEA (0.522 mL, 3 mmol) in DMF (2 mL) wasadded N-Boc-p-phenylenediamine (0.208 g, 1.0 mmol). The reaction mixturewas stirred at room temperature for 0.5 hours and then was concentratedby rotary evaporator. The resulting mixture was treated with 100% TFA atroom temperature for 3 hours to remove the Boc protecting group. Themixture was concentrated again by rotary evaporator, and treated withmethyl oxalyl chloride (0.135 g, 1.1 mmol) and DIEA (0.522 mL, 3 mmol)in DMF (2 mL) for 0.5 hours at room temperature. Finally, the mixturewas hydrolyzed by 10% LiOH to furnish product2-oxo-2-((4-(2-phenyl-2-sulfoacetamido)phenyl)amino)acetic acid (Lib-1).Subsequently, it was subjected to HPLC purification, and product Lib-1was obtained as colorless oil with 56% overall yield and >95% purity.2-oxo-2-((3-(2-phenyl-2-sulfoacetamido)phenyl)amino)acetic acid (Lib-2)and 2-oxo-2-((4-(2-phenyl-2-sulfoacetamido)benzyl)amino)acetic acid(Lib-3) were obtained in similar procedures by usingN-Boc-m-phenylenediamine and 4-[(N-Boc)aminomethyl]aniline,respectively.

2-oxo-2-((4-(2-phenyl-2-sulfoacetamido)phenyl)amino)acetic acid (Lib-1):¹H NMR (500 MHz, DMSO-d6) δ 10.65 (s, 1H), 10.31(s, 1H), 7.69 (d, J=9.1Hz, 2H), 7.57-7.54(m, 4H), 7.30-7.24 (m, 3H), 4.75 (s, 1H). ¹³C NMR (125MHz, DMSO-d6) δ 165.8, 162.2, 156.6, 135.8, 135.3, 133.0, 129.9, 127.4,127.0, 71.8. ESI-MS Cacld. for C16H13N2O7S (M−H⁺): m/z 377.0449; found377.0450.

2-oxo-2-((3-(2-phenyl-2-sulfoacetamido)phenyl)amino)acetic acid (Lib-2):¹H NMR (500 MHz, DMSO-d6) δ 10.68 (s, 1H), 10.36 (s, 1H), 8.03 (t, J=1.6Hz, 1H), 7.57-7.55 (m, 2H), 7.44-7.38 (m, 2H), 7.30-7.24 (m, 4H), 4.78(s, 1H). ¹³C NMR (125 MHz, DMSO-d6) δ 166.0, 162.2, 157.0, 139.4, 138.0,135.2, 129.9, 129.0, 127.5, 127.0, 115.4, 115.3, 111.1, 71.8. ESI-MSCacld. for C16H13N2O7S (M−H⁺): m/z 377.0449; found 377.0445.

2-oxo-2-((4-(2-phenyl-2-sulfoacetamido)benzyl)amino)acetic acid (Lib-3):¹H NMR (500 MHz, DMSO-d6) δ 10.30 (s, 1H), 9.31 (t, J=6.2 Hz, 1H),7.56-7.51 (m, 4H), 7.29-7.19 (m, 5H), 4.74 (s, 1H), 4.26 (d, J=6.3 Hz,2H). ¹³C NMR (125 MHz, DMSO-d6) δ 165.9, 162.2, 158.4, 138.0, 135.3,133.3, 129.9, 127.9, 127.5, 127.0, 118.9, 71.8, 30.7. ESI-MS Cacld. forC17H15N2O7S (M−H⁺): m/z 391.0605; found 391.0609.

2-oxo-2-((4(2-phenyl-2-sulfoacetamido)methyl)phenyl)amino)acetic acid(Lib-4) was synthesized using a slightly different procedure.4-[(N-Boc)aminomethyl]aniline was treated with methyl oxalyl chloride(0.135 g, 1.1 mmol) and DIEA (0.522 mL, 3 mmol) in DMF (2 mL) for 0.5hours at room temperature. The mixture was concentrated and then treatedwith 100% TFA at room temperature for 3 hours to remove the Bocprotecting group. The crude mixture was concentrated by rotaryevaporator and treated with α-sulfophenyl acetyl chloride (0.257 g, 1.1mmol) and DIEA (0.522 mL, 3 mmol) in DMF (2 mL). The mixture was finallyhydrolyzed by 10% LiOH and subjected to HPLC purification, and product2-oxo-2-((4(2-phenyl-2-sulfoacetamido)methyl)phenyl)amino)acetic acid(Lib-4) was obtained as colorless oil in 82% overall yield with >90%purity.

2-oxo-2-((4-(2-phenyl-2-sulfoacetamido)methyl)phenyl)amino)acetic acid(Lib-4): ¹H NMR (500 MHz, DMSO-d6) δ 10.68 (s, 1H), 8.64 (t, J=5.9 Hz,1H), 7.68 (d, J=8.6 Hz, 2H), 7.49-7.48 (m, 2H), 7.29-7.24 (m, 5H), 4.55(s, 1H), 4.31 (dd, J=5.9, 11.1 Hz, 2H). ¹³C NMR (125 MHz, DMSO-d6) δ167.5, 162.2, 156.7, 136.3, 135.6, 135.6, 129.7, 127.4, 127.3, 126.8,120.2, 71.5, 30.7. ESI-MS Cacld. for C17H15N2O7S (M−H⁺): m/z 391.0605;found 391.0599.

SPAA-based library synthesis. To all wells of 96-well plates were added2-oxo-2-((4-(2-phenyl-2-sulfoacetamido)phenyl)amino)acetic acid (Lib-1)(20 μL, 20 mM in DMF), HBTU (20 μL, 20 mM in DMF), HOBt (20 μL, 20 mM inDMF), DIEA (20 μL, 60 mM in DMF), and corresponding 192 amines fromstorage plates (1.2 μL, 0.5 mM in DMF), the reaction plates were placedat room temperature for 1 day. Compounds from2-oxo-2-((3-(2-phenyl-2-sulfoacetamido)phenyl)amino)acetic acid (Lib-2),2-oxo-2-((4-(2-phenyl-2-sulfoacetamido)benzyl)amino)acetic acid (Lib-3)and 2-oxo-2-((4-((2-phenyl-2-sulfoacetamido)methyl)phenyl)amino)aceticacid (Lib-4) were synthesized by the same procedure. The reaction wellsfrom aniline were monitored by LC-MS to show that reactions occurredwell in great conversions. Thus, four libraries of 768 compounds wereobtained with estimated concentration at 4 mM (assuming 80% productyield), which was screened against PTPs as described.

Synthesis of compounds 2-7. To2-oxo-2-((4-(2-phenyl-2-sulfoacetamido)phenyl)amino)acetic acid (Lib-1)(0.0756 g, 0.2 mmol) in DMF (2 mL) was added HBTU (0.078 g, 0.2 mmol),HOBt (0.031 g, 0.2 mmol), DIEA (0.105 mL, 0.6 mmol), and 4-iodoaniline(0.053 g, 0.22 mmol) sequentially, and the reaction mixture was stirredat room temperature for 1 hour. The mixture was then subjected to HPLCpurification, and product2-((4-(2-((4-iodophenyl)amino)-2-oxoacetamido)phenyl)amino)-2-oxo-1-phenylethanesulfonicacid (2) was obtained as colorless oil (97% yield, >95% purity).Products 3 to 7 were obtained in the same manner.

2-((4-(2-((4-iodophenyl)amino)-2-oxoacetamido)phenyl)amino)-2-oxo-1-phenylethanesulfonicacid (2): ¹H NMR (500 MHz, DMSO-d6) δ 10.91 (s, 1H), 10.77 (s, 1H),10.33 (s, 1H), 7.78 (d, J=8.9 Hz, 2H), 7.73-7.68 (m, 4H), 7.60-7.58 (m,4H), 7.30-7.25 (m, 3H), 4.78 (s, 1H). ¹³C NMR (125 MHz, DMSO-d6) δ165.9, 158.9, 158.1, 137.6, 137.4, 135.8, 135.2, 132.8, 129.9, 127.4,126.9, 122.6, 120.9, 119.1, 88.7, 71.8. ESI-MS Cacld. for C22H17IN3O6S(M−H⁺): m/z 577.9888; found 577.9883.

2-oxo-2-((4-(2-oxo-2-((4-(thiophen-2-yl)phenyl)amino)acetamido)phenyl)amino)-1-phenylethanesulfonicacid (3): ¹H NMR (300 MHz, DMSO-d6) δ 10.90 (s, 1H), 10.82(s, 1H), 10.33(s, 1H), 7.93-7.73 (m, 13H), 7.64-7.26 (m, 3H), 4.73 (s, 1H). ESI-MSCacld. for C26H2ON3O6S2 (M−H⁺): m/z 534.0799; found 534.0799.

2-((4-(2-((3-(4-bromophenyl)-1H-pyrazol-5-yl)amino)-2-oxoacetamido)phenyl)amino)-2-oxo-1-phenylethanesulfonicacid (4): ¹H NMR (300 MHz, DMSO-d6) δ 11.00 (s, 1H), 10.80 (s, 1H),10.32 (s, 1H), 7.79-7.54 (m, 10H), 7.28-7.26 (m, 3H), 6.92 (s, 1H), 4.71(s, 1H). ESI-MS Cacld. for C25H19BrN5O6S (M−H⁺): m/z 596.0245; found596.0253.

2-((4-(2-([1,1′-biphenyl]-4-ylamino)-2-oxoacetamido)phenyl)amino)-2-oxo-1-phenylethanesulfonicacid (5): ¹H NMR (300 MHz, DMSO-d6) δ 10.92 (s, 1H), 10.81(s, 1H), 10.31(s, 1H), 7.96-7.26 (m, 18H), 4.72 (s, 1H). ESI-MS Cacld. for C28H22N3O6S(M−H⁺): m/z 528.1235; found 528.1231.

2-((4-(2-((4-isopropylphenyl)amino)-2-oxoacetamido)phenyl)amino)-2-oxo-1-phenylethanesulfonicacid (6): ¹H NMR (300 MHz, DMSO-d6) δ 10.78 (s, 1H), 10.74 (s, 1H),10.33 (s, 1H), 7.80-7.73 (m, 4H), 7.59-7.56 (m, 4H), 7.25-7.22 (m, 5H),4.73 (s, 1H), 2.91-2.82 (m, 1H), 1.19 (d, J=9.0 Hz, 6H). ESI-MS Cacld.for C25H24N3O6S (M−H⁺): m/z 494.1391; found 494.1394.

2-oxo-2-((4-(2-oxo-2-(phenylamino)acetamido)phenyl)amino)-1-phenylethanesulfonicacid (7): ¹H NMR (300 MHz, DMSO-d6) δ 10.80 (s, 2H), 10.34 (s, 1H),7.87-7.78 (m, 4H), 7.60-7.56 (m, 4H), 7.40-7.27 (m, 6H), 4.78 (s, 1H).¹³C NMR (75 MHz, DMSO-d6) δ 166.3, 159.2, 158.8, 138.1, 136.3, 135.6,133.4, 130.3, 129.2, 127.9, 127.4, 125.1, 121.3, 120.9, 119.6, 72.1.ESI-MS Cacld. for C22H18N3O6S (M−H⁺): m/z 452.0922; found 452.0917.

Results and Discussion

Identification and characterization of cefsulodin as a SHP2 Inhibitor.To identify SHP2 inhibitory agents with improved bioavailability, 1,850FDA-approved clinical drugs from the Johns Hopkins Drug Library werescreened against the catalytic domain of SHP2 using a phosphataseactivity-based assay. The drugs were assayed at both 10 and 20 μMconcentrations at 25° C. and pH 7.0 in a buffer containing 50 mM3,3-dimethylglutarate and 1 mM EDTA with an ionic strength of 0.15 Madjusted by addition of NaCl. Positive hits were selected based on adecrease in absorbance that corresponds to a decline in the hydrolysisof the substrate para-nitrophenyl phosphate (pNPP). Cefsulodin(structure shown in FIG. 14), a third generation β-lactam cephalosporinantibiotic with a narrow spectrum restricted to Pseudomonas Aeruginosa,surfaced as a top hit against SHP2. Kinetic measurements withrepurchased samples of cefsulodin confirmed its SHP2 inhibitory activitywith an IC₅₀ value of 16.8±2.0 μM under the assay conditions describedabove. Lineweaver-Burk plot analysis revealed that the mode of SHP2inhibition by cefsulodin is competitive with respect to the substratepNPP with a K_(i) of 6.6±0.2 μM (FIG. 15).

The reactivity of β-lactam ring is important for β-lactam antibiotics'therapeutic activity, but also makes them vulnerable to hydrolysis andattack by various nucleophiles. Since the PTP-catalyzed reaction employsnucleophic catalysis, it was determined whether cefsulodin acts as anirreversible inhibitor and inactivates SHP2 by a covalent mechanism. Tothis end, the IC₅₀ of cefsulodin against SHP2 was re-measured under thesame assay conditions by either incubating cefsulodin with SHP2 for 30minutes prior to the addition of pNPP, or incubating cefsulodin withpNPP for 30 minutes prior to the addition of SHP2. Reversible andfast-binding inhibitors are not expected to display time dependency,whereas irreversible or tight-binding inhibitors will exhibitsignificantly reduced IC₅₀ values when they are pre-incubated with theenzyme. Similar IC₅₀ values were obtained for cefsoludin under bothconditions (cefsoludin pre-mixed with SHP2, IC₅₀=16.8±1.8 μM; cefsoludinpre-mixed with pNPP, IC₅₀=17.5±2.0 μM), suggesting that cefsulodin doesnot inactivate SHP2, at least not within the duration of the assay time.SHP2-catalyzed pNPP hydrolysis in the presence of cefsulodin was alsomonitored by LC-MS as a function of time at the same pH and temperature(FIGS. 16A & 16B). No evidence of SHP2 modification was observed and nochange in cefsulodin concentration was evident.

Finally, SHP2 (100 nM and 10 nM) was incubated with 100 μM cefsulodinfor three hours and the mixtures were analyzed by QTOF ESI-MS (FIGS.17A-17D). The results showed no covalent adduct formation between SHP2and cefsulodin. As a positive control, phenyl vinyl sulfone, a known PTPactivity-based probe that covalently modifies the catalytic cysteine,formed a covalent adduct with SHP2 within 10 minutes (FIG. 17D). Takentogether, these studies indicated that cefsulodin is a competitive andreversible SHP2 inhibitor.

To assess the specificity of cefsulodin for SHP2, its inhibitoryactivity toward a panel of mammalian PTPs, including cytosolic PTPs,SHP1, PTP1B, LYP, HePTP, and PTP-Meg2, receptor-like PTPs, CD45, PTPα,PTPβ, PTPε, PTPγ, PTPμ and LAR, dual specificity phosphatases VHR andCDC14A, low molecular weight (LMW) PTP, and a protein Ser/Thrphosphatase PP5, were measured. As shown in Table 18, cefsulodin isquite selective for SHP2, displaying, with one exception, greater than10-fold selectivity against all PTPs examined The sole exception isSHP1, which exhibits high sequence identity to SHP2. Cefsulodin showssimilar inhibitory activity toward SHP1 with an IC₅₀ of 21±3.0 μM.

TABLE 18 The IC50 values of cefsulodin and novel SHP2 inhibitors againsta panel of PTPs. All measurements were made in a pH 7.0 buffercontaining 50 mM 3,3-dimethylglutarate and 1 mM EDTA with an ionicstrength of 0.15M adjusted by addition of NaCl, using pNPP as substrate.Enzyme Cefsulodin 2 3 4 5 6 SHP2 16.8 ± 2.0 1.51 ± 0.06 0.90 ± 0.1  0.73± 0.02 1.36 ± 0.04 2.33 ± 0.04 SHP1 21.0 ± 3.0 7.33 ± 1.3  2.6 ± 0.04 6.0 ± 0.02 4.8 ± 1.1  6.0 ± 0.02 PTP1B 190 ± 18 9.3 ± 2.0 4.2 ± 1.0  7± 1  3.0 ± 0.06 7 ± 1 LYP >200 >10 >10 >10 >10 >10 HePTP 250 ±20 >10 >10 >10 >10 >10 PTP-Meg2 350 ± 20 >10 >10 >10 >10 >10 CD45 390 ±40 6.6 ± 2  2.5 ± 0.08 6.7 ± 2  3.8 ± 1.0  3.7 ± 0.08PTPα >200 >10 >10 >10 >10 >10 PTPβ >200 >10 >10 >10 >10 >10PTPε >200 >10 >10 >10 >10 >10 PTPγ >200 >10 >10 >10 >10 >10PTPμ >200 >10 >10 >10 >10 >10 LAR >200 >10 >10 >10 >10 >10VHR >200 >10 >10 >10 >10 >10 CDC14A 300 ± 50 >10 >10 >10 >10 >10LMWPTP >200 >10 >10 >10 >10 >10 PP5 >200 >10 >10 >10 >10 >10

To understand the structure and activity relationship ofcefsoludin-mediated SHP2 inhibition, several related β-lactamantibiotics, including moxalactam, carbenicillin, sulbenicillin,cefalonium, cefamandole, cefdinir, cephalexin, and penicillin G, wereacquired. Cefsulodin was dissected into 3 parts: the sulfonic acid headgroup A, the fused β-lactam core B, and the isonicotinamide tail C (FIG.14). Moxalactam is the only compound that possesses all three parts;carbenicillin and sulbenicillin contain parts A and B; cefalonium andcefamandole have parts B and C; and cefdinir, cephalexin and penicillinG have just part B, the β-lactam core. Interestingly, when measured at200 μM compound concentration, moxalactam was the only one thatexhibited modest inhibition against SHP1, with an IC₅₀ of 170 μM. Noneof the other β-lactam antibiotics displayed any inhibition against SHP2at 200 μM, indicating that the structural integrity of cefsulodin isnecessary for SHP2 inhibition.

X-ray crystal structure of SHP2 in complex with cefsulodin andidentification of Sulfo Phenyl Acetic Amide (SPAA) as a novel pTyrmimetic. To define the molecular basis for SHP2 inhibition by cefsulodinand to guide the design of new cefsulodin-based SHP2 inhibitors withimproved potency and selectivity, the crystal structure of SHP2catalytic domain (residues 262-528) in complex with cefsulodin at 1.6 Aresolution was determined. Data collection and structure refinementstatistics are summarized in Table 19. The structure was refined toR_(work)/R_(free) of 17.6%/20.5%. The SHP2●cefsulodin complexcrystallized in the P2₁ space group with one molecule per asymmetricunit. The overall structure is very similar to the reported apo-SHP2structure (PDBID: 3B7O) used for molecular replacement, with an RMSD of0.523 Å for 256 Cα atoms. The presence of a small molecule in SHP2active site was unambiguously identified by the strong positive Fo-Fcelectron density around the catalytic P-loop (FIG. 18A). The smallmolecule binding mode was iteratively built, refined, and confirmed byexcellent 2Fo-Fc electron density contoured at 1.06 (FIG. 18B).

TABLE 19 Data collection and structure refinement statisticsSHP2•Cefsulodin Data Collection Space group P2₁ Cell dimensions a, b, c({acute over (Å)}) 39.64, 75.27, 48.05 α, β, γ (°) 90.0, 99.1, 90.0Resolution ({acute over (Å)}) 1.60 (1.63-1.60) R_(merge) 0.077 (0.666)I/σI 22.1 (1.5) Completeness (%) 97.3 (80.0) Redundancy 3.4 (2.7)Refinement Resolution ({acute over (Å)}) 1.60 No. reflections 35,592R_(work)/R_(tree) 0.176/0.205 No. atoms Protein 2,156 Ligand 29 Water364 R.m.s. deviations Bond lengths ({acute over (Å)}) 0.007 Bond angles(°) 1.183 Ramachandran plot (%) Most favored regions 90.3 Additionalallowed regions 8.0 Generously allowed regions 1.7 Disallowed regions 0The dataset was collected from a single crystal. Values in parenthesesare for highest-resolution shell.

Unexpectedly, the small molecule observed in the SHP2 co-crystalstructure is not the original cefsulodin but an altered form of thedrug: the β-lactam core (part B) is opened, and the isonicotinamidemoiety (part C) is not visible in the structure. This altered form(compound 1, FIG. 18C) is situated in the SHP2 active site with abundantinteractions with the P-loop, pY recognition loop, Q-loop and WPD-loop(FIG. 18D). In detail (FIG. 18E), the sulfonic acid is in closeproximity to the catalytic P-loop and forms multiple hydrogen bonds withthe backbone amides of S460, A461, I463, G464 and R465, as well as twowater-mediated hydrogen bonds with the R465 and K366 side chains. Theα-benzene ring is located within a hydrophobic pocket constituted byA461, I463, I282 and Y279, which normally functions to recognize andstabilize the tyrosine ring of PTP substrates during catalysis.Superimposition of the SHP2●cefsulodin structure with a previouslyreported PTP1B●phosphopeptide structure (PDBID: 1EEN) revealed that theSulfo Phenyl Acetic Amide (SPAA) motif overlaps very well with pTyr inthe phosphopeptide (FIG. 18F). In addition, the newly formed carboxylicacid, as a result of β-lactam ring opening, makes multiple water bridgedpolar interactions with W423 and G427 in the WPD loop as well as T507and Q510 in the Q-loop. This orientation also forces the 6-memberedthiazine ring including the terminal carboxylic acid group to point outof the active site with no obvious interactions with SHP2 residues.

Further inspection of the 2Fo-Fc map at 0.76 revealed weak electrondensity extending from atom X (FIG. 18C) to a space where a flexibleloop (residues 314 to 324), which has not been observed in previouslyreported SHP2 structures, from a symmetry-related SHP2 molecule. Whethera covalent bond was formed between compound 1 and SHP2 was theninvestigated. Thus, co-crystals of the SHP2●cefsulodin complex werecollected, washed and re-dissolved in water, and the resulting solutionwas subjected to QTOF ESI-MS analysis. As a control, apo-SHP2 crystalswere also processed and analyzed in the same way. As shown in FIG. 19A,the re-dissolved apo-SHP2 crystals show only one peak at 32224.57, whichcorresponds to the molecular mass of the SHP2 catalytic domain (residue262-528). In contrast, the re-dissolved co-crystals of SHP2 andcefsulodin had an additional peak at 32652.80 (FIG. 19B), and thedifference between these two peaks is 428.24, which corresponds to themolecular mass of compound 1 minus two hydrogen atoms, likely as aresult of covalent bond formation with SHP2. To identify the residuecovalently attached to compound 1, the missing loop (residue 314 to 324)was re-constructed based on the 2Fo-Fc electron density observed at 0.76through iterative cycles of building and refinement (FIG. 20A). Throughcrystallographic symmetry operations, the atom X was found to overlapnicely with the sulfur atom in C318 from the flexible loop in asymmetry-related SHP2 molecule (FIG. 20B), and the 2Fo-Fc electrondensity at 0.76 adequately accounts for every atom of C318, supportingthe existence of a covalent bond between the SHP2 active site boundcompound 1 and a symmetry-related SHP2 molecule in the co-crystal (FIG.20C).

One plausible explanation for the apparent contradictory findings thatcefsulodin acts as a reversible and competitive SHP2 inhibitor in assaysolution but forms a covalent adduct with SHP2 in the crystalline statemay be that cefsulodin covalently modifies SHP2 during thecrystallization process. Cefsulodin is most stable under pH 4-6 butreadily degrades when the pH is over 7, a process which could beaccelerated by the presence of strong nucleophiles. As mentioned above,there are two reactive sites in cefsulodin (β-lactam core andisonicotinamide), and the loss of isonicotinamide precedes β-lactam ringopening due to higher reactivity.

LC-MS was used to study cefsulodin stability in different buffersolutions (FIGS. 21A-21E). Consistent with previous reports thatcefsulodin stability is highly pH dependent, degradation of cefsulodinwas negligible when monitored in pH 5.8 MES buffer (20 mM MES, 250 mMNaCl, 1 mM EDTA) (FIG. 21B). In contrast, cefsulodin degradation wasobvious in pH 7.4 CBTP buffer (33 mM Citric acid, 67 mM BIS-TRISPropane) (FIG. 21C), and its degradation was further accelerated in CBTPbuffer at pH 9.1 (FIG. 21D).

In the crystallization experiment, the MES (pH 5.8) buffer containing0.2 mM SHP2, 1 mM cefsulodin and 2 mM DTT was mixed with an equal volumeof the CBTP buffer (pH 7.4), and the resulting solution (final pH=7.1)was allowed to stand at 20° C. for several days. Under this condition,cefsulodin was found to be completely degraded within 2 days (FIG. 21E).In addition, conjugate adducts of cefsulodin with either CBTP or DTT(FIGS. 22A and 22B) could be detected in the cefsulodin samples storedin the pH 7.4 CBTP buffer (FIG. 22C) and under crystallizationconditions (FIG. 21E). Given the similar nucleophilicity of the Cys sidechain and DTT, and the proximity of C318 to the isonicotinamide group inthe crystal, it is reasonable to speculate that upon cefsulodin bindingto the SHP2 active site in the crystalline state, the isonicotinamidetail in cefsulodin is poised to be replaced by C318 from a nearbysymmetry-related SHP2 molecule, and the β-lactam ring is subsequentlyopened by water to form the SHP2-compound 1 adduct as observed in thecrystal structure (FIGS. 20A-20C).

Binding mode between cefsulodin and SHP2. Given the observations thatcefsulodin acts as a reversible and competitive inhibitor for SHP2 underassay conditions, further insight into the binding mode between SHP2 andthe intact cefsulodin molecule was obtained using molecular modeling toinvestigate the binding mode between the two molecules. To avoid biasintroduced from the SHP2●compound 1 structure, a previously reportedapo-SHP2 structure (PDB ID: 3B7O) was used for modeling. The top-twobinding modes with indistinguishable calculated binding energies areshown in FIG. 23A. In both cases, cefsulodin binds SHP2 with the SPAAhead group sticking into the active site, similar to what was observedin the SHP2●compound 1 structure. However, the rest of cefsulodin has adifferent binding mode in comparison to that of compound 1. Thisdifference could be explained by the conformational restraints imposedby the intact β-lactam ring, which forces the thiazine pointing awayfrom the SPAA head, making it impossible to form a sandwich-likeintramolecular conformation as observed in the SHP2●compound 1structure. Moreover, the orientation of the isonicotinamide tail in thetwo predicted binding modes are different (FIG. 23A): it either extendsto the Q-loop and WPD-loop in mode I, and/or to the WPD-loop andresidues 360 to 366 in mode II. Specifically, either residue E508 inmode I or R362 in mode II forms polar interactions with the terminalamide of cefsulodin, which serves as an anchor to control theorientation of isonicotinamide tail.

To differentiate and validate the predicted binding modes forcefsulodin, mutants, SHP2/E508A and SHP2/R362A, were generated and theirkinetic parameters, including k_(cat), K_(m) for pNPP and IC₅₀ forcefsulodin, were determined. SHP2/E508A and SHP2/R362A displayed nearlyidentical k_(cat) and K_(m) to those of the wild-type enzyme, indicatingthat neither residue is essential for SHP2 folding and catalysis.Interestingly, point mutation to convert residue E508 to alanine did notaffect inhibition by cefsulodin (IC₅₀ values for wild-type SHP2 andE508A are 16.7±2.0 μM and 19.0±1.0 μM, respectively), whereasreplacement of R362 with alanine increased the IC₅₀ for cefsulodin(46.5±4.9 μM) by 3-fold. These results indicate that R362, not E508,likely participates in binding cefsulodin and suggest that mode II (FIG.23B) may be the preferred SHP2 binding mode for cefsulodin. In thisbinding mode, the isonicotinamide tail is oriented by the rigid β-lactamring and sandwiched by R362 and K364. The carboxylic acid on theβ-lactam ring forms polar interactions with the side chains of K364 andK366. Similar to the observed interactions between SPAA and the activesite in the SHP2●compound 1 structure, the sulfonic acid in cefsulodinis tightly anchored by the P-loop through numerous H-bonds with thebackbone amides, and the α-benzene ring is situated within a hydrophobicpocket formed by residues Y279, I282, A461 and I463. Collectively, theinteraction profiles between SHP2 and compound 1/cefsulodin, as well asstructural comparison with the PTP1B●phosphopeptide complex, identifySPAA as a unique pTyr mimetic, which could be used for the design anddevelopment of novel sulfonic acid based PTP inhibitors.

A structure-guided SPAA fragment-based approach for SHP2 inhibitordevelopment. Given cefsulodin's chemical reactivity and its modestpotency and selectivity for SHP2 (especially vs SHP1), SHP2 inhibitorswith improved chemical stability and inhibitory activity were designedand synthesized based on the structural insights obtained above. Asshown in FIGS. 24A-24C, cefsulodin consists of three parts: part A(SPAA) is essential for SHP2 inhibitory activity as it binds the activesite; part B functions as a linker to connect part C; and part Cinteracts with residues 362-365 in the β₅-β₆ loop. To target both SHP2active site and adjacent less-conserved pockets, SPAA-based compoundlibraries were prepared using appropriately functionalized linkers toreplace the chemically reactive part B and to introduce structuraldiversity (FIG. 24A).

As a proof-of-concept, 4 focused libraries were designed, in which abenzene ring was chosen as the linker, and amide bond formation servedas a vehicle for structural diversification (FIG. 24B). In practice,Lib-1 to Lib-4 were synthesized by coupling the available carboxylicacids on the oxalyl moiety (FIG. 24B) with a set of 192 amines thatdiffer in size, charge, lipophilicity, polarity, solubility, anddrug-like properties (FIGS. 25A & 25B). Lib-1 and Lib-2 differed atwhich position on the benzene ring the oxalic acid is attached to, whileLib-3 and Lib-4 were designed to explore flexibility on either side ofthe benzene linker. The libraries were assembled directly on 96-wellplates by standard HBTU amide coupling chemistry. The quality of thelibraries was ensured by randomly analyzing multiple wells from eachplate by LC-MS, indicating that the reactions went equally well toafford products in excellent yields (70-80%). Thus, a total of 768compounds incorporating the novel pTyr mimetic SPAA were obtained withmolecular weights ranging from 400 to 700. The libraries were screenedat both ˜10 and ˜1 μM against SHP2 in order to shorten the list ofactives.

The top 5 hits (compounds 2 to 6, FIG. 24C) identified from the screenwere re-synthesized, purified, and their IC₅₀ values for SHP2 weredetermined. As shown in Table 20, although precursors Lib-1 to Lib-4showed no inhibitory activity against SHP2 at 50 μM, compounds 2 to 6exhibited IC₅₀ in the range of 0.73-2.33 μM, which are 7-23 fold morepotent than that of cefsulodin. The marked improvement could beexplained by the fact that Lib-1 to Lib-4 lack part C, the terminaldiversity element, which likely interact with residues in the vicinityof the active site. This large difference is also consistent with thefinding that sulbenicillin exhibits no activity against SHP2 (FIG. 14),likely due to the absence of part C as compared to cefsulodin.Interestingly, compounds 2 to 6 are all from Lib-1, with the oxalic acidhandle located at the para position of the benzene linker. In addition,compounds 2 to 6 have either biaryl or single aryl group with bulkysubstituents at the terminal position, indicating a clear preference forhydrophobic moieties in part C. This conclusion is supported by thesubstantial loss of activity when one of the aromatic rings is removedfrom compound 5 (see compound 7 in FIG. 24C and Table 20). Selectivityprofiling against the same panel of protein phosphatases showed thatcompounds 2 and 4 are at least 4- and 8-fold more effective ininhibiting SHP2 against all PTPs tested (Table 18). Furthermore,compounds 2 to 6 also exhibited much greater aqueous stability thancefsulodin (data not shown).

TABLE 20 Inhibitory activities of SPAA-based compounds against SHP2 IC₅₀(μM) SHP2 Lib-1 >50 Lib-2 >50 Lib-3 >50 Lib-4 >50 2 1.51 ± 0.06 3 0.90 ±0.10 4 0.73 ± 0.02 5 1.36 ± 0.04 6 2.33 ± 0.04 7 >50

SPAA-based SHP2 inhibitors block cancer cell growth and SHP2-mediatedsignaling. SHP2 has been recognized as a proto-oncogene, thus smallmolecule SHP2 inhibitors have potential to become new anti-canceragents. It was previously shown that SHP2 is required for the growth ofnon-small cell lung cancer (NSCLC) cell line H1975, which is resistantto EGF receptor inhibitor treatment and harbors secondary gatekeepermutations in the EGF receptor. Knockdown or pharmacological inhibitionof SHP2 blocked H1975 cell proliferation by attenuation of EGF inducedERK1/2 activation. Hence, the cellular efficacy of the SPAA-based SHP2inhibitors in this system were evaluated. As shown in FIG. 26A, compound2, 5, 6 were able to reduce H1975 cell proliferation in a dose dependentmanner, while compound 7 was ineffective as expected, given that it hasno inhibition against SHP2 at 50 μM. In contrast, compounds 3 and 4 hadno significant effect on H1975 cell proliferation, likely due toinefficient cell penetration. Similar phenomenon was also observed withhuman breast cancer cell line MDA-MB-231 (FIG. 26B). Thus, compound 2appeared to be the most efficacious among this series, and it wasselected for further mechanistic study.

Given the requirement of SHP2 activity for EGF induced Ras-ERK1/2pathway activation, the cellular effect of compound 2 was assessed onEGF-induced ERK1/2 activation in H1975 cells. Treatment of H1975 cellswith compound 2 reduced EGF-mediated ERK1/2 phosphorylation in adose-dependent manner, while a structurally related inactive compound 7(IC₅₀>50 μM for SHP2) had no appreciable effect on ERK1/2phosphorylation (FIGS. 26C & 26D). These results are consistent with theobservations from the MTT assay that compound 2 dose dependentlyinhibited H1975 and MDA-MB-231 cell growth while compound 7 did not(FIGS. 26A & 26B).

To provide further evidence that compound 2 blocks cell signalingthrough SHP2 inhibition, compound 2 was analyzed to see if it had anyeffect on PMA (phorbol 12-myristate 13-acetate)-induced ERK1/2activation, which does not require SHP2, but instead involves activationof protein kinase C and Raf in a Ras-independent manner Thus, in thisExample, SHP2 inhibitors are not expected to impact PMA-induced ERK1/2phosphorylation. Indeed, compound 2 had no effect on PMA-induced ERK1/2phosphorylation (FIG. 26E).

Finally, the effect of SHP2 inhibition by compound 2 was evaluated onthe growth of SKBR3 cells in Matrigel. SKBR3 cells are an ERB2 positivebreast cancer cell line that when grown in Matrigel more accuratelyreflect the features of human tumors than when grown on plastic. Thesecells through upregulation of ERB2 strongly activate Ras, which promotesERK1/2 signaling when grown in Matrigel. As shown in FIGS. 27A & 27B,treatment with 20 μM of compound 2 resulted in a partial inhibition ofERK1/2 and cell growth, while at 40 μM of compound 2, ERK1/2 activitywas barely detectable and cell growth was almost completely absent.Taken together, the results showed that compound 2 can specificallyinhibit EGF mediated ERK1/2 activation and the growth of H1975 lungcancer cells, MDA-MB-231 and ErbB2 positive SKBR3 breast cancer cells.

Conclusion

Despite considerable drug discovery efforts devoted to the PTP targetclass, the task of obtaining selective and cell permeable PTP inhibitorsremains highly challenging. As a result, few PTP inhibitors haveprogressed to the clinic. In the present disclosure, a novel drugrepurposing strategy for the discovery of PTP inhibitors with moredrug-like properties is disclosed. In contrast to the prevailingapproach to drug repurposing, which entails identifying new uses forexisting drugs, the present disclosure illustrates another path for drugrepurposing, namely by identifying a successful pharmacophore from anexisting drug for further refinement and design. Since drugs used in theclinic already possess established pharmacokinetic properties andclinical efficacies, the present disclosure identified useful scaffoldsfrom known drugs as starting points for the design of SHP2 inhibitorswith improved pharmacological properties. By screening a largeFDA-approved drug collection, cefsulodin, a third generation β-lactamcephalosporin antibiotic, was found to exhibit SHP2 inhibitory activity.Structural and molecular modeling analyses identify SPAA as a novelnonhydrolyzable pTyr mimetic, which anchors cefsulodin to SHP2 activesite, while interactions of the isonicotinamide tail with residues inthe β₅-β₆ loop enhances cefsulodin's SHP2 binding potency andselectivity. To remove the chemical liability associated with cefsulodinand to transform cefsulodin into more potent and selective SHP2inhibitors, a structure-guided and SPAA fragment-based approach thattargets both the SHP2 active site as well as its surrounding peripheralpockets was employed. This led to the identification of severalSPAA-based SHP2 inhibitors with IC₅₀ in the low to sub-micromolar rangeand several-fold of selectivity against a large panel of mammalian PTPs.Importantly, these SHP2 inhibitors block EGF stimulated ERK1/2activation and exhibit excellent anti-proliferative activity in H1975non-small cell lung cancer, as well as MDA-MB-231 and SKBR3 breastcancer cells, demonstrating the utility of drug repurposing for thedevelopment of PTP inhibitors with more favorable pharmacologicalproperties. Given the obligatory role of SHP2 in growth factor receptormediated signaling, inhibitors of SHP2 will most likely have widespreadutility in cancer treatment.

Example 10

Inhibition of HePTP and LYP. Several compounds in the SPAA based librarydescribed herein (for example, L319-11-M68, L319-13-M68, and L319-16)have been identified as effective inhibitors of HePTP and LYP (Tables 7and 21).

TABLE 21 IC₅₀ values of HePTP and LYP inhibitors against a panel ofPTPs. IC₅₀, μM HePTP LYP LMWPTP SHP2 L319-06-M68 22 >100 >100 >100L319-07-M68 11 >100 >100 >100 L319-08-N58 16 9.9 11 7.28 L319-11-M68 3.27.6 >100 >100 L319-12-M68 3.9 7.43 21 >100 L319-13-M68 4.3 10 27 >100L319-14-M50 25 23 28.8 24 L319-14-M68 4.5 8.3 >100 >100 L319-14-N03 2411.6 >100 74 L319-16-M47 21 22 37 24 L319-16-M60 13 15 18 12 L319-16-M937.6 16 24 16 L319-16-N55 43 35 90 82 L319-16-N58 20 20 32 22 L319-14 2511.48 >100 79 L319-15 21 7.36 >100 70 L319-16 11 5.19 >100 25L319-Br1-N15 0.2 NA NA 13.2 L319-Br1-N47 26.0 NA NA 22.8 L319-Br1-N7621.7 NA NA 9.9

Example 11

Cellular studies of mPTPB inhibitor L319N53. L319N53 was evaluated incells for its in vivo efficacy in mPTPB inhibition. Previously, mPTPBhas been shown to block IFN-γ induced Erk1/2 activation in macrophagecells, while mPTPB inhibitors can rescue this process (Zhou et al, Proc.Natl. Acad. Sci. USA 2010, 107, 4573-4578). As shown in FIG. 28, L319N53increased Erk1/2 phosphorylation in mPTPB transfected Raw264.7 cells atconcentrations of 8 nM, 16 nM, and 32 nM in a dose dependent manner Incontrast, L319N53 has no effect on Erk1/2 phosphorylation in vectorcells. These results indicate an in vivo activity and specificity ofL319N53 to inhibit mPTPB.

Example 12

Cellular studies of LMWPTP inhibitor L335N15. L335N15 was tested for itsefficacy in blocking LMWPTP's activity in vivo. LMWPTP is a negativeregulator of insulin-mediated mitogenic and metabolic signaling (forexample, the IR-PI3K-Akt pathway), although its precise role inregulating insulin action remains unknown. ASO-targeting LMWPTP inhepatocyte and liver cells was able to enhance the phosphorylation andactivity of key insulin signaling intermediates, including insulinreceptor subunit and Akt in response to insulin stimulation.

To assess the effects of L335N15 in insulin signaling pathway, HepG2cells were pretreated with L335N15 for 1 hour and were subsequentlystimulated with 5 nM insulin for 5 minutes. As shown in FIGS. 29A-29C,L335N15 enhanced IR Tyr1162/1163 phosphorylation in a dose-dependentfashion relative to the vehicle DMSO. Consistent with IR activation,downstream Akt phosphorylation (Ser473) was increased. These results,coupled with the observation that L335N15 at 100 μM did not inhibit 24other PTPs, especially those involved in insulin signaling (PTP1B,TcPTP, Meg2, SHP2, PTPα, PTPε, LAR), suggests that the enhanced insulinsignaling is likely due to the specific, in vivo inhibition of LMWPTP byL335N15. Accordingly, LMWPTP inhibitors such as L335N15 described hereincan be used to develop agents for the treatment of type 2 diabetes andinsulin resistance.

In conclusion, novel sulfonic acid based pTyr mimetic compoundsidentified in the present disclosure are effective inhibitors of severaldistinct PTPs, including mpPTPA, mPTPB, LMWPTP, Laforin, SHP2, HePTP,LYP, with unprecedented potency and selectivity. Through a medicinalchemistry effort described herein, specific inhibitors of either LMWPTPor Laforin have been provided. Importantly, the compounds identified inthis disclosure exhibited in vivo activity in increasing Erk1/2phosphorylation (for example, L319N53 inhibition of mPTPB) and insensitizing insulin signaling pathway (for example, L335N15 inhibitionof LMWPTP). These compounds allow for the development of pharmaceuticalformulations for treating diseases associated with abnormal PTPactivities, such as TB, cancer, diabetes, and obesity.

What is claimed is:
 1. A compound of Formula 1a:

or a therapeutically suitable prodrug thereof or a therapeuticallysuitable salt thereof, wherein R₁ is hydrogen and R₂ is C₁-C₁₀ alkyl,aryl, heteroaryl, —NH—R_(2a), —(CH₂)_(m)NH—CO—R_(x), or—(CH2)_(n)-R_(2b)—(CH2)_(q)-NH—CO—CO—NH—R_(y); wherein, when R₂ is arylor heteroaryl, R₂ is optionally substituted with one or more substituentselected from the group consisting of C₁-C₄ alkyl, C₁-C₄ alkoxy, C₁-C₄alkoxy carbonyl, amino, aryl, benzyloxy (—OBn), —CF₃, carboxy, halogen,1-imidazolyl, 4-morpholinyl, and nitro; wherein m, n, and qindependently are 0-4; wherein R_(2a) and R_(2b) independently are aryl;wherein R_(x) and R_(y) independently are aryl or heteroaryl, and thearyl or heteroaryl are independently optionally substituted with one ormore substituent selected from the group consisting of C₁-C₄ alkyl,benzoyl, benzyl, benzyloxy (—OBn), phenyl, halogen,1H-benzimidazole-2-yl, and 2-thiophenyl; or wherein R₁, R₂, and the Natom to which they are attached are joined together to form a monocyclicor bicyclic heterocycle; wherein R₃ is hydrogen or halogen; and whereinR₄ is hydrogen or aryl, the aryl being optionally substituted with oneor more substituent selected from the group consisting of halogen, C₁-C₄alkyl, C₁-C₄ alkoxy, phenyl, nitro, cyano, and —COCF₃.
 2. The compoundof claim 1, wherein R₂ is phenyl or benzo[d]thiazol-2-yl, optionallysubstituted with one or more substituent selected from the groupconsisting of C₁-C₄ alkyl, C₁-C₄ alkoxy, C₁-C₄ alkoxy carbonyl, amino,aryl, benzyloxy (—OBn), —CF₃, carboxy, halogen, 1-imidazolyl,4-morpholinyl, and nitro.
 3. The compound of claim 1, wherein the R₁,R₂, and the N atom to which they are attached are joined together toform a heterocycle selected from the group consisting of:


4. A compound of Formula 2:

wherein R₂′ is heterocycle, optionally substituted with one or moresubstituent selected from the group consisting of C₁-C₄ alkyl, C₁-C₄alkoxy, C₁-C₄ alkoxy carbonyl, amino, aryl, benzyloxy (—OBn), —CF₃,carboxy, halogen, 1-imidazolyl, 4-morpholinyl, and nitro; wherein R₃′ ishydrogen or halogen; and wherein R₄′ is hydrogen or aryl, the aryl beingoptionally substituted with one or more substituent selected from thegroup consisting of halogen, C₁-C₄ alkyl, C₁-C₄ alkoxy, phenyl, nitro,cyano, and —COCF₃.
 5. A method of inhibiting a protein tyrosinephosphatase (PTP) selected from the group consisting of mPTPA, mPTPB,low molecular weight PTP (LMWPTP), and Laforin in a subject in needthereof, the method comprising administering to the subject atherapeutically effective amount of the compound of claim
 1. 6. A methodof treating tuberculosis in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of the compound of claim
 1. 7. A method of treating a cancer in asubject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of the compound of claim 1.8. The method of claim 7 wherein the cancer is selected from the groupconsisting of breast cancer, colon cancer, bladder cancer, and kidneycancer.
 9. A method of treating Lafora disease in a subject in needthereof, the method comprising administering to the subject atherapeutically effective amount of the compound of claim
 1. 10. Amethod of treating type 2 diabetes in a subject in need thereof, themethod comprising administering to the subject a therapeuticallyeffective amount of the compound of claim
 1. 11. A pharmaceuticalcomposition comprising a compound of claim 1 and a pharmaceuticallyacceptable carrier.
 12. A method of inhibiting a protein tyrosinephosphatase (PTP) selected from the group consisting of mPTPA, mPTPB,low molecular weight PTP (LMWPTP), and Laforin in a subject in needthereof, the method comprising administering to the subject atherapeutically effective amount of the compound of claim
 4. 13. Amethod of treating tuberculosis in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of the compound of claim
 4. 14. A method of treating a cancer ina subject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of the compound of claim 4.15. The method of claim 14 wherein the cancer is selected from the groupconsisting of breast cancer, colon cancer, bladder cancer, and kidneycancer.
 16. A method of treating Lafora disease in a subject in needthereof, the method comprising administering to the subject atherapeutically effective amount of the compound of claim
 4. 17. Amethod of treating type 2 diabetes in a subject in need thereof, themethod comprising administering to the subject a therapeuticallyeffective amount of the compound of claim
 4. 18. A pharmaceuticalcomposition comprising a compound of claim 4 and a pharmaceuticallyacceptable carrier.